Phenotypic states become increasingly sensitive to perturbations near a bifurcation in a synthetic gene network

PubMed Central

Axelrod, Kevin; Sanchez, Alvaro; Gore, Jeff

2015-01-01

Microorganisms often exhibit a history-dependent phenotypic response after exposure to a stimulus which can be imperative for proper function. However, cells frequently experience unexpected environmental perturbations that might induce phenotypic switching. How cells maintain phenotypic states in the face of environmental fluctuations remains an open question. Here, we use environmental perturbations to characterize the resilience of phenotypic states in a synthetic gene network near a critical transition. We find that far from the critical transition an environmental perturbation may induce little to no phenotypic switching, whereas close to the critical transition the same perturbation can cause many cells to switch phenotypic states. This loss of resilience was observed for perturbations that interact directly with the gene circuit as well as for a variety of generic perturbations-such as salt, ethanol, or temperature shocks-that alter the state of the cell more broadly. We obtain qualitatively similar findings in natural gene circuits, such as the yeast GAL network. Our findings illustrate how phenotypic memory can become destabilized by environmental variability near a critical transition. DOI: http://dx.doi.org/10.7554/eLife.07935.001 PMID:26302311

A cell culture technique for human epiretinal membranes to describe cell behavior and membrane contraction in vitro.

PubMed

Wertheimer, Christian; Eibl-Lindner, Kirsten H; Compera, Denise; Kueres, Alexander; Wolf, Armin; Docheva, Denitsa; Priglinger, Siegfried G; Priglinger, Claudia; Schumann, Ricarda G

2017-11-01

To introduce a human cell culture technique for investigating in-vitro behavior of primary epiretinal cells and membrane contraction of fibrocellular tissue surgically removed from eyes with idiopathic macular pucker. Human epiretinal membranes were harvested from ten eyes with idiopathic macular pucker during standard vitrectomy. Specimens were fixed on cell culture plastic using small entomological pins to apply horizontal stress to the tissue, and then transferred to standard cell culture conditions. Cell behavior of 400 epiretinal cells from 10 epiretinal membranes was observed in time-lapse microscopy and analyzed in terms of cell migration, cell velocity, and membrane contraction. Immunocytochemistry was performed for cell type-specific antigens. Cell specific differences in migration behavior were observed comprising two phenotypes: (PT1) epiretinal cells moving fast, less directly, with small round phenotype and (PT2) epiretinal cells moving slowly, directly, with elongated large phenotype. No mitosis, no outgrowth and no migration onto the plastic were seen. Horizontal contraction measurements showed variation between specimens. Masses of epiretinal cells with a myofibroblast-like phenotype expressed cytoplasmatic α-SMA stress fibers and correlated with cell behavior characteristics (PT2). Fast moving epiretinal cells (PT1) were identified as microglia by immunostaining. This in-vitro technique using traction application allows for culturing surgically removed epiretinal membranes from eyes with idiopathic macular pucker, demonstrating cell behavior and membrane contraction of primary human epiretinal cells. Our findings emphasize the abundance of myofibroblasts, the presence of microglia and specific differences of cell behavior in these membranes. This technique has the potential to improve the understanding of pathologies at the vitreomacular interface and might be helpful in establishing anti-fibrotic treatment strategies.

Neuropilin-1 interacts with the second branchial arch microenvironment to mediate chick neural crest cell dynamics

PubMed Central

McLennan, Rebecca; Kulesa, Paul M.

2011-01-01

Cranial neural crest cells (NCCs) require neuropilin signaling to reach and invade the branchial arches. Here, we use an in vivo chick model to investigate whether the neuropilin-1 knockdown phenotype is specific to the second branchial arch (ba2), changes in NCC behaviors and phenotypic consequences, and whether neuropilins work together to facilitate entry into and invasion of ba2. We find that cranial NCCs with reduced neuropilin-1 expression displayed shorter protrusions and decreased cell body and nuclear length-to-width ratios characteristic of a loss in polarity and motility, after specific interaction with ba2. Directed NCC migration was rescued by transplantation of transfected cells into rhombomere 4 of younger hosts. Lastly, reduction of neuropilin-2 expression by shRNA either solely or with reduction of neuropilin-1 expression did not lead to a stronger head phenotype. Thus, NCCs, independent of rhombomere origin, require neuropilin-1, but not neuropilin-2 to maintain polarity and directed migration into ba2. PMID:20503363

Direct Observation of Nanoparticle-Cancer Cell Nucleus Interactions

PubMed Central

Dam, Duncan Hieu M.; Lee, Jung Heon; Sisco, Patrick N.; Co, Dick T.; Zhang, Ming; Wasielewski, Michael R.; Odom, Teri W.

2012-01-01

We report the direct visualization of interactions between drug-loaded nanoparticles and the cancer cell nucleus. Nanoconstructs composed of nucleolin-specific aptamers and gold nanostars were actively transported to the nucleus and induced major changes to the nuclear phenotype via nuclear envelope invaginations near the site of the construct. The number of local deformations could be increased by ultra-fast, light-triggered release of the aptamers from the surface of the gold nanostars. Cancer cells with more nuclear envelope folding showed increased caspase 3 and 7 activity (apoptosis) as well as decreased cell viability. This newly revealed correlation between drug-induced changes in nuclear phenotype and increased therapeutic efficacy could provide new insight for nuclear-targeted cancer therapy. PMID:22424173

Stable T-bet(+)GATA-3(+) Th1/Th2 hybrid cells arise in vivo, can develop directly from naive precursors, and limit immunopathologic inflammation.

PubMed

Peine, Michael; Rausch, Sebastian; Helmstetter, Caroline; Fröhlich, Anja; Hegazy, Ahmed N; Kühl, Anja A; Grevelding, Christoph G; Höfer, Thomas; Hartmann, Susanne; Löhning, Max

2013-01-01

Differentiated T helper (Th) cell lineages are thought to emerge from alternative cell fate decisions. However, recent studies indicated that differentiated Th cells can adopt mixed phenotypes during secondary immunological challenges. Here we show that natural primary immune responses against parasites generate bifunctional Th1 and Th2 hybrid cells that co-express the lineage-specifying transcription factors T-bet and GATA-3 and co-produce Th1 and Th2 cytokines. The integration of Th1-promoting interferon (IFN)-γ and interleukin (IL)-12 signals together with Th2-favoring IL-4 signals commits naive Th cells directly and homogeneously to the hybrid Th1/2 phenotype. Specifically, IFN-γ signals are essential for T-bet(+)GATA-3(+) cells to develop in vitro and in vivo by breaking the dominance of IL-4 over IL-12 signals. The hybrid Th1/2 phenotype is stably maintained in memory cells in vivo for months. It resists reprogramming into classic Th1 or Th2 cells by Th1- or Th2-promoting stimuli, which rather induce quantitative modulations of the combined Th1 and Th2 programs without abolishing either. The hybrid phenotype is associated with intermediate manifestations of both Th1 and Th2 cell properties. Consistently, hybrid Th1/2 cells support inflammatory type-1 and type-2 immune responses but cause less immunopathology than Th1 and Th2 cells, respectively. Thus, we propose the self-limitation of effector T cells based on the stable cell-intrinsic balance of two opposing differentiation programs as a novel concept of how the immune system can prevent excessive inflammation.

Effect of culture medium on propagation and phenotype of corneal stroma-derived stem cells.

PubMed

Sidney, Laura E; Branch, Matthew J; Dua, Harminder S; Hopkinson, Andrew

2015-12-01

The limbal area of the corneal stroma has been identified as a source of mesenchymal-like stem cells, which have potential for exploitation as a cell therapy. However, the optimal culture conditions are disputed and few direct media comparisons have been performed. In this report, we evaluated several media types to identify the optimal for inducing an in vitro stem cell phenotype. Primary human corneal stroma-derived stem cells (CSSCs) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult; serum-free keratinocyte medium (K-SFM); and StemPro-34. Effects on proliferation, morphology, protein and messenger RNA expression were evaluated. All media supported proliferation of CSSCs with the exception of K-SFM and StemPro-34. Morphology differed between media: DMEM produced large cells, whereas EGM produced very small cells. Culture in M199 produced a typical mesenchymal stromal cell phenotype with high expression of CD105, CD90 and CD73 but not CD34. Culture in SCM produced a phenotype more reminiscent of a progenitor cell type with expression of CD34, ABCG2, SSEA-4 and PAX6. Culture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we consider it to be the most appropriate for development as a clinical-grade medium for the production of CSSC phenotypes suitable for cell therapy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Matrix Metalloproteinase Stromelysin-1 Triggers a Cascade of Molecular Alterations that leads to stable epithelial-to-Mesenchymal Conversion and a Premalignant Phenotype in Mammary Epithelial Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lochter, A.; Galosy, S.; Muschler, J.

1997-08-11

Matrix metalloproteinases (MMPs) regulate ductal morphogenesis, apoptosis, and neoplastic progression in mammary epithelial cells. To elucidate the direct effects of MMPs on mammary epithelium, we generated functionally normal cells expressing an inducible autoactivating stromelysin-1 (SL-1) transgene. Induction of SL-1 expression resulted in cleavage of E-cadherin, and triggered progressive phenotypic conversion characterized by disappearance of E-cadherin and catenins from cell-cell contacts, downregulation of cytokeratins, upregulation of vimentin, induction of keratinocyte growth factor expression and activation, and upregulation of endogenous MMPs. Cells expressing SL-1 were unable to undergo lactogenic differentiation and became invasive. Once initiated, this phenotypic conversion was essentially stable, andmore » progressed even in the absence of continued SL-1 expression. These observations demonstrate that inappropriate expression of SL-1 initiates a cascade of events that may represent a coordinated program leading to loss of the differentiated epithelial phenotype and gain of some characteristics of tumor cells. Our data provide novel insights into how MMPs function in development and neoplastic conversion.« less

FGFR1 promotes the stem cell-like phenotype of FGFR1-amplified non-small cell lung cancer cells through the Hedgehog pathway.

PubMed

Ji, Wenxiang; Yu, Yongfeng; Li, Ziming; Wang, Guan; Li, Fan; Xia, Weiliang; Lu, Shun

2016-03-22

Cancer stem cell-like phenotype is critical for tumor formation and treatment resistance. FGFR1 is found to be amplified in non-small cell lung cancer, particularly in the lung squamous cell cancer (LSCC). Whether FGFR1 contributes to the maintenance of stem cell-like phenotype of FGFR1-amplified lung cancer cells remains elusive. In this study, treatment with FGFR1 inhibitor AZD4547 suppressed the growth of tumor spheres and reduced ALDH positive proportion in FGFR1-amplified lung cancer cells in vitro, as well as inhibited the growth of oncospheres and parental cells in xenograft models. Knockdown of FGFR1 recaptured the similar effect as AZD4547 in vitro. Furthermore, activation of FGFR1 and subsequently its downstream ERK signaling enhanced the expression and transcriptional activity of GLI2, which could be blocked by FGFR1 inhibitor/silencing or ERK inhibitor. Knockdown of GLI2 directly inhibited the stem-like phenotype of FGFR1-amilified cells, whereas overexpression of GLI2 sufficiently rescued the phenotype caused by FGFR1 knockdown. Notably we also identified a correlation between FGFR1 and GLI2 expressions from clinical data, as well as an inverse relationship with progression free survival (PFS). Together our study suggests that the FGFR1/GLI2 axis promotes the lung cancer stem cell-like phenotype. These results support a rational strategy of combination of FGFR1 and GLI inhibitors for treatment of FGFR1-amplified lung cancers, especially LSCC.

Phenotypic and metabolic traits of commercial Saccharomyces cerevisiae yeasts

PubMed Central

2014-01-01

Currently, pursuing yeast strains that display both a high potential fitness for alcoholic fermentation and a favorable impact on quality is a major goal in the alcoholic beverage industry. This considerable industrial interest has led to many studies characterizing the phenotypic and metabolic traits of commercial yeast populations. In this study, 20 Saccharomyces cerevisiae strains from different geographical origins exhibited high phenotypic diversity when their response to nine biotechnologically relevant conditions was examined. Next, the fermentation fitness and metabolic traits of eight selected strains with a unique phenotypic profile were evaluated in a high-sugar synthetic medium under two nitrogen regimes. Although the strains exhibited significant differences in nitrogen requirements and utilization rates, a direct relationship between nitrogen consumption, specific growth rate, cell biomass, cell viability, acetic acid and glycerol formation was only observed under high-nitrogen conditions. In contrast, the strains produced more succinic acid under the low-nitrogen regime, and a direct relationship with the final cell biomass was established. Glucose and fructose utilization patterns depended on both yeast strain and nitrogen availability. For low-nitrogen fermentation, three strains did not fully degrade the fructose. This study validates phenotypic and metabolic diversity among commercial wine yeasts and contributes new findings on the relationship between nitrogen availability, yeast cell growth and sugar utilization. We suggest that measuring nitrogen during the stationary growth phase is important because yeast cells fermentative activity is not exclusively related to population size, as previously assumed, but it is also related to the quantity of nitrogen consumed during this growth phase. PMID:24949272

Phenotypic and metabolic traits of commercial Saccharomyces cerevisiae yeasts.

PubMed

Barbosa, Catarina; Lage, Patrícia; Vilela, Alice; Mendes-Faia, Arlete; Mendes-Ferreira, Ana

2014-01-01

Currently, pursuing yeast strains that display both a high potential fitness for alcoholic fermentation and a favorable impact on quality is a major goal in the alcoholic beverage industry. This considerable industrial interest has led to many studies characterizing the phenotypic and metabolic traits of commercial yeast populations. In this study, 20 Saccharomyces cerevisiae strains from different geographical origins exhibited high phenotypic diversity when their response to nine biotechnologically relevant conditions was examined. Next, the fermentation fitness and metabolic traits of eight selected strains with a unique phenotypic profile were evaluated in a high-sugar synthetic medium under two nitrogen regimes. Although the strains exhibited significant differences in nitrogen requirements and utilization rates, a direct relationship between nitrogen consumption, specific growth rate, cell biomass, cell viability, acetic acid and glycerol formation was only observed under high-nitrogen conditions. In contrast, the strains produced more succinic acid under the low-nitrogen regime, and a direct relationship with the final cell biomass was established. Glucose and fructose utilization patterns depended on both yeast strain and nitrogen availability. For low-nitrogen fermentation, three strains did not fully degrade the fructose. This study validates phenotypic and metabolic diversity among commercial wine yeasts and contributes new findings on the relationship between nitrogen availability, yeast cell growth and sugar utilization. We suggest that measuring nitrogen during the stationary growth phase is important because yeast cells fermentative activity is not exclusively related to population size, as previously assumed, but it is also related to the quantity of nitrogen consumed during this growth phase.

Chromatin organization as an indicator of glucocorticoid induced natural killer cell dysfunction.

PubMed

Misale, Michael S; Witek Janusek, Linda; Tell, Dina; Mathews, Herbert L

2018-01-01

It is well-established that psychological distress reduces natural killer cell immune function and that this reduction can be due to the stress-induced release of glucocorticoids. Glucocorticoids are known to alter epigenetic marks associated with immune effector loci, and are also known to influence chromatin organization. The purpose of this investigation was to assess the effect of glucocorticoids on natural killer cell chromatin organization and to determine the relationship of chromatin organization to natural killer cell effector function, e.g. interferon gamma production. Interferon gamma production is the prototypic cytokine produced by natural killer cells and is known to modulate both innate and adaptive immunity. Glucocorticoid treatment of human peripheral blood mononuclear cells resulted in a significant reduction in interferon gamma production. Glucocorticoid treatment also resulted in a demonstrable natural killer cell nuclear phenotype. This phenotype was localization of the histone, post-translational epigenetic mark, H3K27me3, to the nuclear periphery. Peripheral nuclear localization of H3K27me3 was directly related to cellular levels of interferon gamma. This nuclear phenotype was determined by direct visual inspection and by use of an automated, high through-put technology, the Amnis ImageStream. This technology combines the per-cell information content provided by standard microscopy with the statistical significance afforded by large sample sizes common to standard flow cytometry. Most importantly, this technology provides for a direct assessment of the localization of signal intensity within individual cells. The results demonstrate glucocorticoids to dysregulate natural killer cell function at least in part through altered H3K27me3 nuclear organization and demonstrate H3K27me3 chromatin organization to be a predictive indicator of glucocorticoid induced immune dysregulation of natural killer cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Human somatic cells subjected to genetic induction with six germ line-related factors display meiotic germ cell-like features

PubMed Central

Medrano, Jose V.; Martínez-Arroyo, Ana M.; Míguez, Jose M.; Moreno, Inmaculada; Martínez, Sebastián; Quiñonero, Alicia; Díaz-Gimeno, Patricia; Marqués-Marí, Ana I.; Pellicer, Antonio; Remohí, Jose; Simón, Carlos

2016-01-01

The in vitro derivation of human germ cells has attracted interest in the last years, but their direct conversion from human somatic cells has not yet been reported. Here we tested the ability of human male somatic cells to directly convert into a meiotic germ cell-like phenotype by inducing them with a combination of selected key germ cell developmental factors. We started with a pool of 12 candidates that were reduced to 6, demonstrating that ectopic expression of the germ line-related genes PRDM1, PRDM14, LIN28A, DAZL, VASA and SYCP3 induced direct conversion of somatic cells (hFSK (46, XY), and hMSC (46, XY)) into a germ cell-like phenotype in vitro. Induced germ cell-like cells showed a marked switch in their transcriptomic profile and expressed several post-meiotic germ line related markers, showed meiotic progression, evidence of epigenetic reprogramming, and approximately 1% were able to complete meiosis as demonstrated by their haploid status and the expression of several post-meiotic markers. Furthermore, xenotransplantation assays demonstrated that a subset of induced cells properly colonize the spermatogonial niche. Knowledge obtained from this work can be used to create in vitro models to study gamete-related diseases in humans. PMID:27112843

Epigenetic modulation of the miR-200 family is associated with transition to a breast cancer stem-cell-like state.

PubMed

Lim, Yat-Yuen; Wright, Josephine A; Attema, Joanne L; Gregory, Philip A; Bert, Andrew G; Smith, Eric; Thomas, Daniel; Lopez, Angel F; Drew, Paul A; Khew-Goodall, Yeesim; Goodall, Gregory J

2013-05-15

The miR-200 family is a key regulator of the epithelial-mesenchymal transition, however, its role in controlling the transition between cancer stem-cell-like and non-stem-cell-like phenotypes is not well understood. We utilized immortalized human mammary epithelial (HMLE) cells to investigate the regulation of the miR-200 family during their conversion to a stem-like phenotype. HMLE cells were found to be capable of spontaneous conversion from a non-stem to a stem-like phenotype and this conversion was accompanied by the loss of miR-200 expression. Stem-like cell fractions isolated from metastatic breast cancers also displayed loss of miR-200 indicating similar molecular changes may occur during breast cancer progression. The phenotypic change observed in HMLE cells was directly controlled by miR-200 because restoration of its expression decreased stem-like properties while promoting a transition to an epithelial phenotype. Investigation of the mechanisms controlling miR-200 expression revealed both DNA methylation and histone modifications were significantly altered in the stem-like and non-stem phenotypes. In particular, in the stem-like phenotype, the miR-200b-200a-429 cluster was silenced primarily through polycomb group-mediated histone modifications whereas the miR-200c-141 cluster was repressed by DNA methylation. These results indicate that the miR-200 family plays a crucial role in the transition between stem-like and non-stem phenotypes and that distinct epigenetic-based mechanisms regulate each miR-200 gene in this process. Therapy targeted against miR-200 family members and epigenetic modifications might therefore be applicable to breast cancer.

Engineering live cell surfaces with functional polymers via cytocompatible controlled radical polymerization

NASA Astrophysics Data System (ADS)

Niu, Jia; Lunn, David J.; Pusuluri, Anusha; Yoo, Justin I.; O'Malley, Michelle A.; Mitragotri, Samir; Soh, H. Tom; Hawker, Craig J.

2017-06-01

The capability to graft synthetic polymers onto the surfaces of live cells offers the potential to manipulate and control their phenotype and underlying cellular processes. Conventional grafting-to strategies for conjugating preformed polymers to cell surfaces are limited by low polymer grafting efficiency. Here we report an alternative grafting-from strategy for directly engineering the surfaces of live yeast and mammalian cells through cell surface-initiated controlled radical polymerization. By developing cytocompatible PET-RAFT (photoinduced electron transfer-reversible addition-fragmentation chain-transfer polymerization), synthetic polymers with narrow polydispersity (Mw/Mn < 1.3) could be obtained at room temperature in 5 minutes. This polymerization strategy enables chain growth to be initiated directly from chain-transfer agents anchored on the surface of live cells using either covalent attachment or non-covalent insertion, while maintaining high cell viability. Compared with conventional grafting-to approaches, these methods significantly improve the efficiency of grafting polymer chains and enable the active manipulation of cellular phenotypes.

Cryopreservation has no effect on function of natural killer cells differentiated in vitro from umbilical cord blood CD34(+) cells.

PubMed

Domogala, Anna; Madrigal, J Alejandro; Saudemont, Aurore

2016-06-01

Natural killer (NK) cells offer the potential for a powerful cellular immunotherapy because they can target malignant cells without being direct effectors of graft-versus-host disease. We have previously shown that high numbers of functional NK cells can be differentiated in vitro from umbilical cord blood (CB) CD34(+) cells. To develop a readily available, off-the-shelf cellular product, it is essential that NK cells differentiated in vitro can be frozen and thawed while maintaining the same phenotype and functions. We evaluated the phenotype and function of fresh and frozen NK cells differentiated in vitro. We also assessed whether the concentration of NK cells at the time of freezing had an impact on cell viability. We found that cell concentration of NK cells at the time of freezing did not have an impact on their viability and on cell recovery post-thaw. Moreover, freezing of differentiated NK cells in vitro did not affect their phenotype, cytotoxicity and degranulation capacity toward K562 cells, cytokine production and proliferation. We are therefore able to generate large numbers of functional NK cells from CB CD34(+) cells that maintain the same phenotype and function post-cryopreservation, which will allow for multiple infusions of a highly cytotoxic NK cell product. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Incorporation of Biomaterials in Multicellular Aggregates Modulates Pluripotent Stem Cell Differentiation

PubMed Central

Bratt-Leal, Andrés M.; Carpenedo, Richard L.; Ungrin, Mark; Zandstra, Peter W.; McDevitt, Todd C.

2010-01-01

Biomaterials are increasingly being used to engineer the biochemical and biophysical properties of the extracellular stem cell microenvironment in order to tailor niche characteristics and direct cell phenotype. To date, stem cell-biomaterial interactions have largely been studied by introducing stem cells into artificial environments, such as 2D cell culture on biomaterial surfaces, encapsulation of cell suspensions within hydrogel materials, or cell seeding on 3D polymeric scaffolds. In this study, microparticles fabricated from different materials, such as agarose, PLGA and gelatin, were stably integrated, in a dose-dependent manner, within aggregates of pluripotent stem cells (PSCs) prior to differentiation as a means to directly examine stem cell-biomaterial interactions in 3D. Interestingly, the presence of the materials within the stem cell aggregates differentially modulated the gene and protein expression patterns of several differentiation markers without adversely affecting cell viability. Microparticle incorporation within 3D stem cell aggregates can control the spatial presentation of extracellular environmental cues (i.e. soluble factors, extracellular matrix and intercellular adhesion molecules) as a means to direct the differentiation of stem cells for tissue engineering and regenerative medicine applications. In addition, these results suggest that the physical presence of microparticles within stem cell aggregates does not compromise PSC differentiation, but in fact the choice of biomaterials can impact the propensity of stem cells to adopt particular differentiated cell phenotypes. PMID:20864164

Cytomegalovirus Infection Leads to Development of High Frequencies of Cytotoxic Virus-Specific CD4+ T Cells Targeted to Vascular Endothelium

PubMed Central

Begum, Jusnara; Lal, Neeraj; Zuo, Jianmin; Beggs, Andrew; Moss, Paul

2016-01-01

Cytomegalovirus (CMV) infection elicits a very strong and sustained intravascular T cell immune response which may contribute towards development of accelerated immune senescence and vascular disease in older people. Virus-specific CD8+ T cell responses have been investigated extensively through the use of HLA-peptide tetramers but much less is known regarding CMV-specific CD4+ T cells. We used a range of HLA class II-peptide tetramers to investigate the phenotypic and transcriptional profile of CMV-specific CD4+ T cells within healthy donors. We show that such cells comprise an average of 0.45% of the CD4+ T cell pool and can reach up to 24% in some individuals (range 0.01–24%). CMV-specific CD4+ T cells display a highly differentiated effector memory phenotype and express a range of cytokines, dominated by dual TNF-α and IFN-γ expression, although substantial populations which express IL-4 were seen in some donors. Microarray analysis and phenotypic expression revealed a profile of unique features. These include the expression of CX3CR1, which would direct cells towards fractalkine on activated endothelium, and the β2-adrenergic receptor, which could permit rapid response to stress. CMV-specific CD4+ T cells display an intense cytotoxic profile with high level expression of granzyme B and perforin, a pattern which increases further during aging. In addition CMV-specific CD4+ T cells demonstrate strong cytotoxic activity against antigen-loaded target cells when isolated directly ex vivo. PD-1 expression is present on 47% of cells but both the intensity and distribution of the inhibitory receptor is reduced in older people. These findings reveal the marked accumulation and unique phenotype of CMV-specific CD4+ T cells and indicate how such T cells may contribute to the vascular complications associated with CMV in older people. PMID:27606804

Mitochondria and the evolutionary roots of cancer

NASA Astrophysics Data System (ADS)

Davila, Alfonso F.; Zamorano, Pedro

2013-04-01

Cancer disease is inherent to, and widespread among, metazoans. Yet, some of the hallmarks of cancer such as uncontrolled cell proliferation, lack of apoptosis, hypoxia, fermentative metabolism and free cell motility (metastasis) are akin to a prokaryotic lifestyle, suggesting a link between cancer disease and evolution. In this hypothesis paper, we propose that cancer cells represent a phenotypic reversion to the earliest stage of eukaryotic evolution. This reversion is triggered by the dysregulation of the mitochondria due to cumulative oxidative damage to mitochondrial and nuclear DNA. As a result, the phenotype of normal, differentiated cells gradually reverts to the phenotype of a facultative anaerobic, heterotrophic cell optimized for survival and proliferation in hypoxic environments. This phenotype matches the phenotype of the last eukaryotic common ancestor (LECA) that resulted from the endosymbiosis between an α-proteobacteria (which later became the mitochondria) and an archaebacteria. As such, the evolution of cancer within one individual can be viewed as a recapitulation of the evolution of the eukaryotic cell from fully differentiated cells to LECA. This evolutionary model of cancer is compatible with the current understanding of the disease, and explains the evolutionary basis for most of the hallmarks of cancer, as well as the link between the disease and aging. It could also open new avenues for treatment directed at reestablishing the synergy between the mitochondria and the cancerous cell.

Versican and the regulation of cell phenotype in disease.

PubMed

Wight, Thomas N; Kinsella, Michael G; Evanko, Stephen P; Potter-Perigo, Susan; Merrilees, Mervyn J

2014-08-01

Versican is an extracellular matrix (ECM) proteoglycan that is present in the pericellular environment of most tissues and increases in many different diseases. Versican interacts with cells to influence the ability of cells to proliferate, migrate, adhere and assemble an ECM. The structure of the versican molecule is briefly reviewed and studies highlighting those factors that promote versican synthesis and degradation and their impact on cell phenotype in disease are discussed. Particular attention is given to vascular disease, but other diseases where versican is important are covered as well, most notably different forms of cancers. Attention is given to mechanisms(s) by which versican influences cell behaviors through either direct or indirect processes. Versican produced by either stromal cells or myeloid cells can have a major impact influencing immunity and inflammation. Finally, studies controlling versican accumulation that either delay or inhibit the progression of disease will be highlighted. Versican is one component of the ECM that can influence the ability of cells to proliferate, migrate, adhere, and remodel the ECM. Targeting versican as a way to control cell phenotype offers a novel approach in the treatment of disease. ECM molecules such as versican contribute to the structural integrity of tissues and interact with cells through direct and indirect means to regulate, in part, cellular events that form the basis of disease. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. Copyright © 2014 Elsevier B.V. All rights reserved.

Adult pituitary stem cells: from pituitary plasticity to adenoma development.

PubMed

Florio, Tullio

2011-01-01

The pituitary needs high plasticity of the hormone-producing cell compartment to generate the continuously changing hormonal signals that govern the key physiological processes it is involved in, as well as homeostatic cell turnover. However, the underlying mechanisms are still poorly understood. It was proposed that adult stem cells direct the generation of newborn cells with a hormonal phenotype according to the physiological requirements. However, only in recent years adult pituitary stem cells have begun to be phenotypically characterized in several studies that identified multiple stem/progenitor cell candidates. Also considering the incompletely defined features of this cell subpopulation, some discrepancies among the different reports are clearly apparent and long-term self-renewal remains to be unequivocally demonstrated. Here, all the recently published evidence is analyzed, trying, when possible, to reconcile the results of the different studies. Finally, with the perspective of shedding light on pituitary tumorigenesis and the development of potentially new pharmacological approaches directed against these cells, very recent evidence on the presence of putative cancer stem cells in human pituitary adenomas is discussed. Copyright © 2011 S. Karger AG, Basel.

Direct phenotypic and genotypic detection of a recombinant pseudomonad population released into lake water.

PubMed Central

Morgan, J A; Winstanley, C; Pickup, R W; Jones, J G; Saunders, J R

1989-01-01

As a system for studying the fate of genetically engineered microorganisms in the environment, we have previously constructed recombinant plasmids encoding a xylE marker gene (C. Winstanley, J. A. W. Morgan, R. W. Pickup, J. G. Jones, and J. R. Saunders, Appl. Environ. Microbiol. 55:771-777, 1989). A series of direct membrane filter methods have been developed which facilitate the detection of bacterial cells harboring the xylE gene, its product, catechol 2,3-dioxygenase, and catechol 2,3-dioxygenase enzyme activity directly from water samples. These methods enable detection of recombinant populations at concentrations as low as 10(3) to 10(4) cells ml of lake water-1. Direct detection facilitates ecological studies of a range of bacterial strains containing the marker system in aquatic environments. The fate of a recombinant pseudomonad population in lake water was assessed by a combination of colony-forming ability, direct counts, and direct detection of the xylE gene and phenotypic expression of its product. Images PMID:2604395

Nanotopography guides and directs cell migration in amoeboid and epithelial cells

NASA Astrophysics Data System (ADS)

Lee, Rachel; Das, Satarupa; Hourwitz, Matthew; Sun, Xiaoyu; Parent, Carole; Fourkas, John; Losert, Wolfgang

Cell migration plays a critical role in development, angiogenesis, immune response, wound healing, and cancer metastasis. In many cases, cells also move in the context of a matrix of collagen fibers, and the alignment of these fibers can both affect the migration phenotype and guide cells. Here we show that both fast and slow migrating cells - amoeboid HL-60 and epithelial MCF10A - are affected in similar ways by micro/nanostructures with dimensions similar to those of collagen fibers. Cell alignment enhances the efficiency of migration by increasing directional persistence.

Canine mammary cancer cells direct macrophages toward an intermediate activation state between M1/M2.

PubMed

Beirão, Breno C B; Raposo, Teresa; Pang, Lisa Y; Argyle, David J

2015-07-15

Canine mammary carcinoma is the most common cancer in female dogs and is often fatal due to the development of distance metastasis. The microenvironment of a tumour often contains abundant infiltrates of macrophages called tumour-associated macrophages (TAMs). TAMs express an activated phenotype, termed M2, which sustains proliferation of cancer cells, and has been correlated with poor clinical outcomes in human cancer patients. Cancer cells themselves have been implicated in stimulating the conversion of macrophages to a TAM with an M2 phenotype. This process has yet to be fully elucidated. Here we investigate the interplay between cancer cells and macrophages in the context of canine mammary carcinoma. We show that cancer cells inhibit lipopolysaccharide (LPS)-induced macrophage activation. Further, we show that macrophage associated proteins, colony-stimulating factor (CSF)-1 and C-C motif ligand (CCL)-2, stimulate macrophages and are responsible for the effects of cancer cells on macrophages. We suggest the existence of a feedback loop between macrophages and cancer cells; while cancer cells influence the phenotype of the TAMs through CSF-1 and CCL2, the macrophages induce canine mammary cancer cells to upregulate their own expression of the receptors for CSF-1 and CCL2 and increase the cancer cellular metabolic activity. However, these cytokines in isolation induce a phenotypic state in macrophages that is between M1 and M2 phenotypes. Overall, our results demonstrate the extent to which canine mammary carcinoma cells influence the macrophage phenotype and the relevance of a feedback loop between these cells, involving CSF-1 and CCL2 as important mediators.

Virus-specific CD4+ memory phenotype T cells are abundant in unexposed adults

PubMed Central

Su, Laura F.; Kidd, Brian A.; Han, Arnold; Kotzin, Jonathan J.; Davis, Mark M.

2013-01-01

While T cell memory is generally thought to require direct antigen exposure, we find an abundance of memory phenotype cells (20–90%, averaging over 50%) of CD4+ T cells specific for viral antigens in adults that have never been infected. These cells express the appropriate memory markers and genes, rapidly produce cytokines, and have clonally expanded. This contrasts with newborns where the same T cell receptor (TCR) specificities are almost entirely naïve, which may explain the vulnerability of young children to infections. One mechanism for this phenomenon is TCR cross-reactivity to environmental antigens and in support of this we find extensive cross-recognition by HIV-1 and influenza-reactive T lymphocytes to other microbial peptides and the expansion of one of these following influenza vaccination. Thus the presence of these memory phenotype T cells has significant implications for immunity to novel pathogens, child and adult health, and the influence of pathogen-rich versus hygienic environments. PMID:23395677

Mesenchymal Stem Cell Spheroids Retain Osteogenic Phenotype Through α2β1 Signaling

PubMed Central

Murphy, Kaitlin C.; Hoch, Allison I.; Harvestine, Jenna N.; Zhou, Dejie

2016-01-01

The induction of mesenchymal stem cells (MSCs) toward the osteoblastic lineage using osteogenic supplements prior to implantation is one approach under examination to enhance their bone-forming potential. MSCs rapidly lose their induced phenotype upon removal of the soluble stimuli; however, their bone-forming potential can be sustained when provided with continued instruction via extracellular matrix (ECM) cues. In comparison with dissociated cells, MSC spheroids exhibit improved survival and secretion of trophic factors while maintaining their osteogenic potential. We hypothesized that entrapment of MSC spheroids formed from osteogenically induced cells would exhibit better preservation of their bone-forming potential than would dissociated cells from monolayer culture. Spheroids exhibited comparable osteogenic potential and increased proangiogenic potential with or without osteogenic preconditioning versus monolayer-cultured MSCs. Spheroids were then entrapped in collagen hydrogels, and the osteogenic stimulus was removed. In comparison with entrapped dissociated MSCs, spheroids exhibited significantly increased markers of osteogenic differentiation. The capacity of MSC spheroids to retain their osteogenic phenotype upon withdrawal of inductive cues was mediated by α2β1 integrin binding to cell-secreted ECM. These results demonstrate the capacity of spheroidal culture to sustain the mineral-producing phenotype of MSCs, thus enhancing their contribution toward bone formation and repair. Significance Despite the promise of mesenchymal stem cells (MSCs) for cell-based therapies for tissue repair and regeneration, there is little evidence that transplanted MSCs directly contribute to new bone formation, suggesting that induced cells rapidly lose their osteogenic phenotype or undergo apoptosis. In comparison with dissociated cells, MSC spheroids exhibit increased trophic factor secretion and improved cell survival. The loss of phenotype represents a significant clinical challenge for cell therapies, yet there is no evidence for whether MSC spheroids retain their osteogenic phenotype upon entrapment in a clinically relevant biomaterial. These findings demonstrate that MSC spheroids retain their osteogenic phenotype better than do dissociated MSCs, and this is due to integrin engagement with the cell-secreted extracellular matrix. These data provide evidence for a novel approach for potentiating the use of MSCs in bone repair. PMID:27365484

Cross-species functional analysis of cancer-associated fibroblasts identifies a critical role for CLCF1 and IL6 in non-small cell lung cancer in vivo

PubMed Central

Vicent, Silvestre; Sayles, Leanne C.; Vaka, Dedeepya; Khatri, Purvesh; Gevaert, Olivier; Chen, Ron; Zheng, Yanyan; Gillespie, Anna K.; Clarke, Nicole; Xu, Yue; Shrager, Joseph; Hoang, Chuong D.; Plevritis, Sylvia; Butte, Atul J.; Sweet-Cordero, E. Alejandro

2013-01-01

Cancer-associated fibroblasts (CAFs) have been reported to support tumor progression by a variety of mechanisms. However, their role in the progression of non-small cell lung cancer (NSCLC) remains poorly defined. In addition, the extent to which specific proteins secreted by CAFs contribute directly to tumor growth is unclear. To study the role of CAFs in NSCLC, a cross-species functional characterization of mouse and human lung CAFs was performed. CAFs supported the growth of lung cancer cells in vivo by secretion of soluble factors that directly stimulate the growth of tumor cells. Gene expression analysis comparing normal mouse lung fibroblasts (NFs) and mouse lung CAFs identified multiple genes that correlate with the CAF phenotype. A gene signature of secreted genes upregulated in CAFs was an independent marker of poor survival in NSCLC patients. This secreted gene signature was upregulated in NFs after long-term exposure to tumor cells, demonstrating that NFs are “educated” by tumor cells to acquire a CAF-like phenotype. Functional studies identified important roles for CLCF1-CNTFR and IL6-IL6R signaling, in promoting growth of NSCLC cells. This study identifies novel soluble factors contributing to the CAF protumorigenic phenotype in NSCLC and suggests new avenues for the development of therapeutic strategies. PMID:22962265

Translation of Genotype to Phenotype by a Hierarchy of Cell Subsystems.

PubMed

Yu, Michael Ku; Kramer, Michael; Dutkowski, Janusz; Srivas, Rohith; Licon, Katherine; Kreisberg, Jason; Ng, Cherie T; Krogan, Nevan; Sharan, Roded; Ideker, Trey

2016-02-24

Accurately translating genotype to phenotype requires accounting for the functional impact of genetic variation at many biological scales. Here we present a strategy for genotype-phenotype reasoning based on existing knowledge of cellular subsystems. These subsystems and their hierarchical organization are defined by the Gene Ontology or a complementary ontology inferred directly from previously published datasets. Guided by the ontology's hierarchical structure, we organize genotype data into an "ontotype," that is, a hierarchy of perturbations representing the effects of genetic variation at multiple cellular scales. The ontotype is then interpreted using logical rules generated by machine learning to predict phenotype. This approach substantially outperforms previous, non-hierarchical methods for translating yeast genotype to cell growth phenotype, and it accurately predicts the growth outcomes of two new screens of 2,503 double gene knockouts impacting DNA repair or nuclear lumen. Ontotypes also generalize to larger knockout combinations, setting the stage for interpreting the complex genetics of disease.

Phenotypic convergence in bacterial adaptive evolution to ethanol stress.

PubMed

Horinouchi, Takaaki; Suzuki, Shingo; Hirasawa, Takashi; Ono, Naoaki; Yomo, Tetsuya; Shimizu, Hiroshi; Furusawa, Chikara

2015-09-03

Bacterial cells have a remarkable ability to adapt to environmental changes, a phenomenon known as adaptive evolution. During adaptive evolution, phenotype and genotype dynamically changes; however, the relationship between these changes and associated constraints is yet to be fully elucidated. In this study, we analyzed phenotypic and genotypic changes in Escherichia coli cells during adaptive evolution to ethanol stress. Phenotypic changes were quantified by transcriptome and metabolome analyses and were similar among independently evolved ethanol tolerant populations, which indicate the existence of evolutionary constraints in the dynamics of adaptive evolution. Furthermore, the contribution of identified mutations in one of the tolerant strains was evaluated using site-directed mutagenesis. The result demonstrated that the introduction of all identified mutations cannot fully explain the observed tolerance in the tolerant strain. The results demonstrated that the convergence of adaptive phenotypic changes and diverse genotypic changes, which suggested that the phenotype-genotype mapping is complex. The integration of transcriptome and genome data provides a quantitative understanding of evolutionary constraints.

Correction of xeroderma pigmentosum complementation group D mutant cell phenotypes by chromosome and gene transfer: Involvement of the human ERCC2 DNA repair gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Flejter, W.L.; McDaniel, L.D.; Johns, D.

1992-01-01

Cultured cells from individuals afflicted with the genetically heterogeneous autosomal recessive disorder xeroderma pigmentosum (XP) exhibit sensitivity to UV radiation and defective nucleotide excision repair. Complementation of these mutant phenotypes after the introduction of single human chromosomes from repair-proficient cells into XP cells has provided a means of mapping the genes involved in this disease. The authors now report the phenotypic correction of XP cells from genetic complementation group D (XP-D) by a single human chromosome designated Tneo. Detailed molecular characterization of Tneo revealed a rearranged structure involving human chromosomes 16 and 19, including the excision repair cross-complementing 2 (ERCC2)more » gene from the previously described human DNA repair gene cluster at 19q13.2-q13.3. Direct transfer of a cosmid bearing the ERCC2 gene conferred UV resistance to XP-D cells.« less

Nuclear EGFR-PKM2 axis induces cancer stem cell-like characteristics in irradiation-resistant cells.

PubMed

Shi, Ying; Liu, Na; Lai, Weiwei; Yan, Bin; Chen, Ling; Liu, Shouping; Liu, Shuang; Wang, Xiang; Xiao, Desheng; Liu, Xiaoli; Mao, Chao; Jiang, Yiqun; Jia, Jiantao; Liu, Yating; Yang, Rui; Cao, Ya; Tao, Yongguang

2018-05-28

Radiation therapy has become an important tool in the treatment of cancer patients, but most patients relapse within 5 years. Relapse is due to the presence of cancer stem cells (CSCs), but the molecular mechanism of radioresistance in CSCs remains largely elusive. Here, we found that irradiation-resistant (IR) cells exhibited increased stem cell-like properties together with elevated anchorage-independent growth and metastasis ability. EGFR not only leads to increased acquisition of endometrial cancer stem cell markers in radioresistant sublines but is critical for the cancer stem-cell phenotype and tumorigenicity. Moreover, PKM2 functions as an interacting partner of EGFR, which induces the EMT phenotype and stem cell-like properties in IR cells. Finally, we found that the regulatory function of the EGFR-PKM2 axis is dependent on nuclear EGFR. In sum, our study indicated that EGFR and PKM2 directly interact and bind with each other to regulate the transcription of stemness-related genes and promote the stem-like phenotype, thus promoting invasion and metastasis. Copyright © 2018 Elsevier B.V. All rights reserved.

Suppression of endogenous lipogenesis induces reversion of the malignant phenotype and normalized differentiation in breast cancer

PubMed Central

Schroeder, Barbara; Park, Cheol Hong; Chandra Mohan, KVP; Khurana, Ashwani; Corominas-Faja, Bruna; Cuyàs, Elisabet; Alarcón, Tomás; Kleer, Celina; Menendez, Javier A.; Lupu, Ruth

2016-01-01

The correction of specific signaling defects can reverse the oncogenic phenotype of tumor cells by acting in a dominant manner over the cancer genome. Unfortunately, there have been very few successful attempts at identifying the primary cues that could redirect malignant tissues to a normal phenotype. Here we show that suppression of the lipogenic enzyme fatty acid synthase (FASN) leads to stable reversion of the malignant phenotype and normalizes differentiation in a model of breast cancer (BC) progression. FASN knockdown dramatically reduced tumorigenicity of BC cells and restored tissue architecture, which was reminiscent of normal ductal-like structures in the mammary gland. Loss of FASN signaling was sufficient to direct tumors to a reversed phenotype that was near normal when considering the development of polarized growth-arrested acinar-like structure similar to those formed by nonmalignant breast cells in a 3D reconstituted basement membrane in vitro. This process, in vivo, resulted in a low proliferation index, mesenchymal-epithelial transition, and shut-off of the angiogenic switch in FASN-depleted BC cells orthotopically implanted into mammary fat pads. The role of FASN as a negative regulator of correct breast tissue architecture and terminal epithelial cell differentiation was dominant over the malignant phenotype of tumor cells possessing multiple cancer-driving genetic lesions as it remained stable during the course of serial in vivo passage of orthotopic tumor-derived cells. Transient knockdown of FASN suppressed hallmark structural and cytosolic/secretive proteins (vimentin, N-cadherin, fibronectin) in a model of EMT-induced cancer stem cells (CSC). Indirect pharmacological inhibition of FASN promoted a phenotypic switch from basal- to luminal-like tumorsphere architectures with reduced intrasphere heterogeneity. The fact that sole correction of exacerbated lipogenesis can stably reprogram cancer cells back to normal-like tissue architectures might open a new avenue to chronically restrain BC progression by using FASN-based differentiation therapies. PMID:27223424

Defective interleukin-4/Stat6 activity correlates with increased constitutive expression of negative regulators SOCS-3, SOCS-7, and CISH in colon cancer cells.

PubMed

Liu, Xiao Hong; Xu, Shuang Bing; Yuan, Jia; Li, Ben Hui; Zhang, Yan; Yuan, Qin; Li, Pin Dong; Li, Feng; Zhang, Wen Jie

2009-12-01

Interleukin-4 (IL-4)-induced Stat6 activities (phenotypes) vary among human cancer cells, of which the HT-29 cell line carries an active Stat6(high) phenotype, while Caco-2 carries a defective Stat6(null) phenotype, respectively. Cancer cells with Stat6(high) show resistance to apoptosis and exaggerated metastasis, suggesting the clinical significance of Stat6 phenotypes. We previously showed that Stat6(high) HT-29 cells exhibited low constitutive expression of Stat6-negative regulators SOCS-1 and SHP-1 because of gene hypermethylation. This study further examined the constitutive expression of other closely related SOCS family numbers including SOCS-3, SOCS-5, SOCS-7, and CISH using RT-PCR. Similar to SOCS-1 and SHP-1, Stat6(high) HT-29 cells expressed low constitutive mRNA of SOCS-3, SOCS-7, and CISH than Stat6(null) Caco-2 cells. Interestingly, DNA demethylation using 5-aza-2'-deoxycytidine in HT-29 cells up-regulated mRNA expression of the above genes, indicating a hypermethylation status, which was confirmed by methylation-specific sequencing in selected SOCS-3 gene. Furthermore, defective Stat6(null) Caco-2 exhibited impaired phosphorylation of Stat6 after IL-4 stimulation by flow cytometry, in keeping with the notion of an over-performed negative regulation. The findings that IL-4/Stat6 phenotypes show differential expression of multiple negative regulators suggest a model that a collective force of powerful negative regulators, directly and indirectly, acts on Stat6 activation, which may result in differential Stat6 phenotypes.

Mechanical regulation of stem-cell differentiation by the stretch-activated Piezo channel.

PubMed

He, Li; Si, Guangwei; Huang, Jiuhong; Samuel, Aravinthan D T; Perrimon, Norbert

2018-03-01

Somatic stem cells constantly adjust their self-renewal and lineage commitment by integrating various environmental cues to maintain tissue homeostasis. Although numerous chemical and biological signals have been identified that regulate stem-cell behaviour, whether stem cells can directly sense mechanical signals in vivo remains unclear. Here we show that mechanical stress regulates stem-cell differentiation in the adult Drosophila midgut through the stretch-activated ion channel Piezo. We find that Piezo is specifically expressed in previously unidentified enteroendocrine precursor cells, which have reduced proliferation ability and are destined to become enteroendocrine cells. Loss of Piezo activity reduces the generation of enteroendocrine cells in the adult midgut. In addition, ectopic expression of Piezo in all stem cells triggers both cell proliferation and enteroendocrine cell differentiation. Both the Piezo mutant and overexpression phenotypes can be rescued by manipulation of cytosolic Ca 2+ levels, and increases in cytosolic Ca 2+ resemble the Piezo overexpression phenotype, suggesting that Piezo functions through Ca 2+ signalling. Further studies suggest that Ca 2+ signalling promotes stem-cell proliferation and differentiation through separate pathways. Finally, Piezo is required for both mechanical activation of stem cells in a gut expansion assay and the increase of cytosolic Ca 2+ in response to direct mechanical stimulus in a gut compression assay. Thus, our study demonstrates the existence of a specific group of stem cells in the fly midgut that can directly sense mechanical signals through Piezo.

Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mizoshiri, N.; Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto; Kishida, T.

Introduction: Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. Materials and Methods: We examined which Oct family genesmore » and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. Results: As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. Discussion: The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases. - Highlights: • Introducing L-myc in a combination with either Oct3/4, Oct6 or Oct9 enables the conversion of fibroblasts to osteoblasts. • A combination of L-myc with Oct3/4 or Oct9 can induce the cells to a phenotype closer to normal osteoblasts. • N-myc was considered the most appropriate Myc family gene for induction of osteoblast-like phenotype in fibroblasts. • The combination of Oct9 plus N-myc has the strongest capability of inducing osteoblast-like phenotype.« less

Oncogenic ALK regulates EMT in non-small cell lung carcinoma through repression of the epithelial splicing regulatory protein 1.

PubMed

Voena, Claudia; Varesio, Lydia M; Zhang, Liye; Menotti, Matteo; Poggio, Teresa; Panizza, Elena; Wang, Qi; Minero, Valerio G; Fagoonee, Sharmila; Compagno, Mara; Altruda, Fiorella; Monti, Stefano; Chiarle, Roberto

2016-05-31

A subset of Non-Small Cell Lung Carcinoma (NSCLC) carries chromosomal rearrangements involving the Anaplastic Lymphoma Kinase (ALK) gene. ALK-rearranged NSCLC are typically adenocarcinoma characterized by a solid signet-ring cell pattern that is frequently associated with a metastatic phenotype. Recent reports linked the presence of ALK rearrangement to an epithelial-mesenchymal transition (EMT) phenotype in NSCLC, but the extent and the mechanisms of an ALK-mediated EMT in ALK-rearranged NSCLC are largely unknown. We found that the ALK-rearranged H2228 and DFCI032, but not the H3122, cell lines displayed a mesenchymal phenotype. In these cell lines, oncogenic ALK activity dictated an EMT phenotype by directly suppressing E-cadherin and up-regulating vimentin expression, as well as expression of other genes involved in EMT. We found that the epithelial splicing regulatory protein 1 (ESRP1), a key regulator of the splicing switch during EMT, was repressed by EML4-ALK activity. The treatment of NSCLC cells with ALK tyrosine kinase inhibitors (TKIs) led to up-regulation of ESRP1 and E-cadherin, thus reverting the phenotype from mesenchymal to epithelial (MET). Consistently, ESRP1 knock-down impaired E-cadherin up-regulation upon ALK inhibition, whereas enforced expression of ESRP1 was sufficient to increase E-cadherin expression. These findings demonstrate an ALK oncogenic activity in the regulation of an EMT phenotype in a subset of NSCLC with potential implications for the biology of ALK-rearranged NSCLC in terms of metastatic propensity and resistance to therapy.

Oncogenic ALK regulates EMT in non-small cell lung carcinoma through repression of the epithelial splicing regulatory protein 1

PubMed Central

Menotti, Matteo; Poggio, Teresa; Panizza, Elena; Wang, Qi; Minero, Valerio G.; Fagoonee, Sharmila; Compagno, Mara; Altruda, Fiorella; Monti, Stefano; Chiarle, Roberto

2016-01-01

A subset of Non-Small Cell Lung Carcinoma (NSCLC) carries chromosomal rearrangements involving the Anaplastic Lymphoma Kinase (ALK) gene. ALK-rearranged NSCLC are typically adenocarcinoma characterized by a solid signet-ring cell pattern that is frequently associated with a metastatic phenotype. Recent reports linked the presence of ALK rearrangement to an epithelial-mesenchymal transition (EMT) phenotype in NSCLC, but the extent and the mechanisms of an ALK-mediated EMT in ALK-rearranged NSCLC are largely unknown. We found that the ALK-rearranged H2228 and DFCI032, but not the H3122, cell lines displayed a mesenchymal phenotype. In these cell lines, oncogenic ALK activity dictated an EMT phenotype by directly suppressing E-cadherin and up-regulating vimentin expression, as well as expression of other genes involved in EMT. We found that the epithelial splicing regulatory protein 1 (ESRP1), a key regulator of the splicing switch during EMT, was repressed by EML4-ALK activity. The treatment of NSCLC cells with ALK tyrosine kinase inhibitors (TKIs) led to up-regulation of ESRP1 and E-cadherin, thus reverting the phenotype from mesenchymal to epithelial (MET). Consistently, ESRP1 knock-down impaired E-cadherin up-regulation upon ALK inhibition, whereas enforced expression of ESRP1 was sufficient to increase E-cadherin expression. These findings demonstrate an ALK oncogenic activity in the regulation of an EMT phenotype in a subset of NSCLC with potential implications for the biology of ALK-rearranged NSCLC in terms of metastatic propensity and resistance to therapy. PMID:27119231

Dynamic Interactions Between Cancer Stem Cells And Their Stromal Partners.

PubMed

Park, Tea Soon; Donnenberg, Vera S; Donnenberg, Albert D; Zambidis, Elias T; Zimmerlin, Ludovic

2014-03-01

The cancer stem cell (CSC) paradigm presumes the existence of self-renewing cancer cells capable of regenerating all tumor compartments and exhibiting stem cell-associated phenotypes. Recent interpretations of the CSC hypothesis envision stemness as a dynamic trait of tumor-initiating cells rather than a defined and unique cell type. Bidirectional crosstalk between the tumor microenvironment and the cancer bulk is well described in the literature and the tumor-associated stroma, vasculature and immune infiltrate have all been implicated as direct contributors to tumor development. These non-neoplastic cell types have also been shown to organize specific niches within the tumor bulk where they can control the intra-tumor CSC content and alter the fate of CSCs and tumor progenitors during tumorigenesis to acquire phenotypic features for invasion, metastasis and dormancy. Despite the complexity of the tumor-stroma interactome, novel therapeutic approaches envision combining tumor-ablative treatment with manipulation of the tumor microenvironment. We will review the currently available literature that provides clues about the complex cellular network that regulate the CSC phenotype and its niches during tumor progression.

Phenotypic and functional characterization of circulating polyomavirus BK VP1-specific CD8+ T cells in healthy adults.

PubMed

van Aalderen, Michiel C; Remmerswaal, Ester B M; Heutinck, Kirstin M; ten Brinke, Anja; Pircher, Hanspeter; van Lier, René A W; ten Berge, Ineke J M

2013-09-01

The human polyomavirus BK virus (BKV) establishes a latent and asymptomatic infection in the majority of the population. In immunocompromised individuals, the virus frequently (re)activates and may cause severe disease such as interstitial nephritis and hemorrhagic cystitis. Currently, the therapeutic options are limited to reconstitution of the antiviral immune response. T cells are particularly important for controlling this virus, and T cell therapies may provide a highly specific and effective mode of treatment. However, little is known about the phenotype and function of BKV-specific T cells in healthy individuals. Using tetrameric BKV peptide-HLA-A02 complexes, we determined the presence, phenotype, and functional characteristics of circulating BKV VP1-specific CD8(+) T cells in 5 healthy individuals. We show that these cells are present in low frequencies in the circulation and that they have a resting CD45RA(-) CD27(+) memory and predominantly CCR7(-) CD127(+) KLRG1(+) CD49d(hi) CXCR3(hi) T-bet(int) Eomesodermin(lo) phenotype. Furthermore, their direct cytotoxic capacity seems to be limited, since they do not readily express granzyme B and express only little granzyme K. We compared these cells to circulating CD8(+) T cells specific for cytomegalovirus (CMV), Epstein-Barr virus (EBV), and influenza virus (Flu) in the same donors and show that BKV-specific T cells have a phenotype that is distinct from that of CMV- and EBV-specific T cells. Lastly, we show that BKV-specific T cells are polyfunctional since they are able to rapidly express interleukin-2 (IL-2), gamma interferon (IFN-γ), tumor necrosis factor α, and also, to a much lower extent, MIP-1β and CD107a.

Phenotypic and Functional Characterization of Circulating Polyomavirus BK VP1-Specific CD8+ T Cells in Healthy Adults

PubMed Central

Remmerswaal, Ester B. M.; Heutinck, Kirstin M.; ten Brinke, Anja; Pircher, Hanspeter; van Lier, René A. W.; ten Berge, Ineke J. M.

2013-01-01

The human polyomavirus BK virus (BKV) establishes a latent and asymptomatic infection in the majority of the population. In immunocompromised individuals, the virus frequently (re)activates and may cause severe disease such as interstitial nephritis and hemorrhagic cystitis. Currently, the therapeutic options are limited to reconstitution of the antiviral immune response. T cells are particularly important for controlling this virus, and T cell therapies may provide a highly specific and effective mode of treatment. However, little is known about the phenotype and function of BKV-specific T cells in healthy individuals. Using tetrameric BKV peptide-HLA-A02 complexes, we determined the presence, phenotype, and functional characteristics of circulating BKV VP1-specific CD8+ T cells in 5 healthy individuals. We show that these cells are present in low frequencies in the circulation and that they have a resting CD45RA− CD27+ memory and predominantly CCR7− CD127+ KLRG1+ CD49dhi CXCR3hi T-betint Eomesoderminlo phenotype. Furthermore, their direct cytotoxic capacity seems to be limited, since they do not readily express granzyme B and express only little granzyme K. We compared these cells to circulating CD8+ T cells specific for cytomegalovirus (CMV), Epstein-Barr virus (EBV), and influenza virus (Flu) in the same donors and show that BKV-specific T cells have a phenotype that is distinct from that of CMV- and EBV-specific T cells. Lastly, we show that BKV-specific T cells are polyfunctional since they are able to rapidly express interleukin-2 (IL-2), gamma interferon (IFN-γ), tumor necrosis factor α, and also, to a much lower extent, MIP-1β and CD107a. PMID:23864628

Toxoplasma gondii-infected natural killer cells display a hypermotility phenotype in vivo.

PubMed

Ueno, Norikiyo; Lodoen, Melissa B; Hickey, Graeme L; Robey, Ellen A; Coombes, Janine L

2015-01-01

Toxoplasma gondii is a highly prevalent intracellular protozoan parasite that causes severe disease in congenitally infected or immunocompromised hosts. T. gondii is capable of invading immune cells and it has been suggested that the parasite harnesses the migratory pathways of these cells to spread through the body. Although in vitro evidence suggests that the parasite further enhances its spread by inducing a hypermotility phenotype in parasitized immune cells, in vivo evidence for this phenomenon is scarce. Here we use a physiologically relevant oral model of T. gondii infection, in conjunction with two-photon laser scanning microscopy, to address this issue. We found that a small proportion of natural killer (NK) cells in mesenteric lymph nodes contained parasites. Compared with uninfected 'bystander' NK cells, these infected NK cells showed faster, more directed and more persistent migratory behavior. Consistent with this, infected NK cells showed impaired spreading and clustering of the integrin, LFA-1, when exposed to plated ligands. Our results provide the first evidence for a hypermigratory phenotype in T. gondii-infected NK cells in vivo, providing an anatomical context for understanding how the parasite manipulates immune cell motility to spread through the host.

Stem Cell Microencapsulation for Phenotypic Control, Bioprocessing, and Transplantation

PubMed Central

Wilson, Jenna L.

2014-01-01

Cell microencapsulation has been utilized for decades as a means to shield cells from the external environment while simultaneously permitting transport of oxygen, nutrients, and secretory molecules. In designing cell therapies, donor primary cells are often difficult to obtain and expand to appropriate numbers, rendering stem cells an attractive alternative due to their capacities for self-renewal, differentiation, and trophic factor secretion. Microencapsulation of stem cells offers several benefits, namely the creation of a defined microenvironment which can be designed to modulate stem cell phenotype, protection from hydrodynamic forces and prevention of agglomeration during expansion in suspension bioreactors, and a means to transplant cells behind a semi-permeable barrier, allowing for molecular secretion while avoiding immune reaction. This review will provide an overview of relevant microencapsulation processes and characterization in the context of maintaining stem cell potency, directing differentiation, investigating scalable production methods, and transplanting stem cells for clinically relevant disorders. PMID:23239279

Genetic Modifiers of Sickle Cell Disease

PubMed Central

Steinberg, Martin H.; Sebastiani, Paola

2015-01-01

Sickle cell anemia is associated with unusual clinical heterogeneity for a Mendelian disorder. Fetal hemoglobin concentration and coincident ∝ thalassemia, both which directly affect the sickle erythrocyte, are the major modulators of the phenotype of disease. Understanding the genetics underlying the heritable subphenotypes of sickle cell anemia would be prognostically useful, could inform personalized therapeutics, and might help the discovery of new “druggable” pathophysiologic targets. Genotype-phenotype association studies have been used to identify novel genetic modifiers. In the future, whole genome sequencing with its promise of discovering hitherto unsuspected variants could add to our understanding of the genetic modifiers of this disease. PMID:22641398

Cell signaling directing the formation and function of hemogenic endothelium during murine embryogenesis

USDA-ARS?s Scientific Manuscript database

During developmental hematopoiesis, multilineage hematopoietic progenitors are thought to derive from a subset of vascular endothelium. Herein, we define the phenotype of such hemogenic endothelial cells and demonstrate, on a clonal level, that they exhibit multilineage hematopoietic potential. Furt...

Polymer microarray technology for stem cell engineering

PubMed Central

Coyle, Robert; Jia, Jia; Mei, Ying

2015-01-01

Stem cells hold remarkable promise for applications in tissue engineering and disease modeling. During the past decade, significant progress has been made in developing soluble factors (e.g., small molecules and growth factors) to direct stem cells into a desired phenotype. However, the current lack of suitable synthetic materials to regulate stem cell activity has limited the realization of the enormous potential of stem cells. This can be attributed to a large number of materials properties (e.g., chemical structures and physical properties of materials) that can affect stem cell fate. This makes it challenging to design biomaterials to direct stem cell behavior. To address this, polymer microarray technology has been developed to rapidly identify materials for a variety of stem cell applications. In this article, we summarize recent developments in polymer array technology and their applications in stem cell engineering. Statement of significance Stem cells hold remarkable promise for applications in tissue engineering and disease modeling. In the last decade, significant progress has been made in developing chemically defined media to direct stem cells into a desired phenotype. However, the current lack of the suitable synthetic materials to regulate stem cell activities has been limiting the realization of the potential of stem cells. This can be attributed to the number of variables in material properties (e.g., chemical structures and physical properties) that can affect stem cells. Polymer microarray technology has shown to be a powerful tool to rapidly identify materials for a variety of stem cell applications. Here we summarize recent developments in polymer array technology and their applications in stem cell engineering. PMID:26497624

Phenotypic correction of Fanconi anemia cells in the murine bone marrow after carrier cell mediated delivery of lentiviral vector.

PubMed

Chakkaramakkil Verghese, Santhosh; Goloviznina, Natalya A; Kurre, Peter

2016-11-19

Fanconi anemia (FA) is an autosomal-recessive disorder associated with hematopoietic failure and it is a candidate for hematopoietic stem cell (HSC)-directed gene therapy. However, the characteristically reduced HSC numbers found in FA patients, their ineffective mobilization from the marrow, and re-oxygenation damage during ex vivo manipulation have precluded clinical success using conventional in vitro approaches. We previously demonstrated that lentiviral vector (LV) particles reversibly attach to the cell surface where they gain protection from serum complement neutralization. We reasoned that cellular delivery of LV to the bone marrow niche could avoid detrimental losses during FA HSC mobilization and in vitro modification. Here, we demonstrate that a VSV-G pseudotyped lentivector, carrying the FANCC transgene, can be transmitted from carrier to bystander cells. In cell culture and transplantation models of FA, we further demonstrate that LV carrier cells migrate along SDF-1α gradients and transfer vector particles that stably integrate and phenotypically correct the characteristic DNA alkylator sensitivity in murine and human FA-deficient target bystander cells. Altogether, we demonstrate that cellular homing mechanisms can be harnessed for the functional phenotype correction in murine FA hematopoietic cells.

Could the Extended Phenotype Extend to the Cellular and Subcellular Levels in Insect-Induced Galls?

PubMed Central

Carneiro, Renê Gonçalves da Silva; Pacheco, Priscilla; Isaias, Rosy Mary dos Santos

2015-01-01

Neo-ontogenesis of plant galls involves redifferentiation of host plant tissues to express new phenotypes, when new cell properties are established via structural-functional remodeling. Herein, Psidium cattleianum leaves and Nothotrioza cattleiani galls are analyzed by developmental anatomy, cytometry and immunocytochemistry of cell walls. We address hypothesis-driven questions concerning the organogenesis of globoid galls in the association of P. cattleianum - N. cattleianum, and P. myrtoides - N. myrtoidis. These double co-generic systems represent good models for comparing final gall shapes and cell lineages functionalities under the perspective of convergent plant-dependent or divergent insect-induced characteristics. Gall induction, and growth and development are similar in both galls, but homologous cell lineages exhibit divergent degrees of cell hypertrophy and directions of elongation. Median cortical cells in P. cattleianum galls hypertrophy the most, while in P. myrtoides galls there is a centrifugal gradient of cell hypertrophy. Cortical cells in P. cattleianum galls tend to anisotropy, while P. myrtoidis galls have isotropically hypertrophied cells. Immunocytochemistry evidences the chemical identity and functional traits of cell lineages: epidermal cells walls have homogalacturonans (HGAs) and galactans, which confer rigidity to sites of enhanced cell division; oil gland cell walls have arabinogalactan proteins (AGPs) that help avoiding cell death; and parenchyma cell walls have HGAs, galactans and arabinans, which confer porosity. Variations in such chemical identities are related to specific sites of hypertrophy. Even though the double co-generic models have the same macroscopic phenotype, the globoid morphotype, current analyses indicate that the extended phenotype of N. cattleiani is substantiated by cellular and subcellular specificities. PMID:26053863

Stable subcutaneous cartilage regeneration of bone marrow stromal cells directed by chondrocyte sheet.

PubMed

Li, Dan; Zhu, Lian; Liu, Yu; Yin, Zongqi; Liu, Yi; Liu, Fangjun; He, Aijuan; Feng, Shaoqing; Zhang, Yixin; Zhang, Zhiyong; Zhang, Wenjie; Liu, Wei; Cao, Yilin; Zhou, Guangdong

2017-05-01

In vivo niche plays an important role in regulating differentiation fate of stem cells. Due to lack of proper chondrogenic niche, stable cartilage regeneration of bone marrow stromal cells (BMSCs) in subcutaneous environments is always a great challenge. This study explored the feasibility that chondrocyte sheet created chondrogenic niche retained chondrogenic phenotype of BMSC engineered cartilage (BEC) in subcutaneous environments. Porcine BMSCs were seeded into biodegradable scaffolds followed by 4weeks of chondrogenic induction in vitro to form BEC, which were wrapped with chondrocyte sheets (Sheet group), acellular small intestinal submucosa (SIS, SIS group), or nothing (Blank group) respectively and then implanted subcutaneously into nude mice to trace the maintenance of chondrogenic phenotype. The results showed that all the constructs in Sheet group displayed typical cartilaginous features with abundant lacunae and cartilage specific matrices deposition. These samples became more mature with prolonged in vivo implantation, and few signs of ossification were observed at all time points except for one sample that had not been wrapped completely. Cell labeling results in Sheet group further revealed that the implanted BEC directly participated in cartilage formation. Samples in both SIS and Blank groups mainly showed ossified tissue at all time points with partial fibrogenesis in a few samples. These results suggested that chondrocyte sheet could create a chondrogenic niche for retaining chondrogenic phenotype of BEC in subcutaneous environment and thus provide a novel research model for stable ectopic cartilage regeneration based on stem cells. In vivo niche plays an important role in directing differentiation fate of stem cells. Due to lack of proper chondrogenic niche, stable cartilage regeneration of bone marrow stromal cells (BMSCs) in subcutaneous environments is always a great challenge. The current study demonstrated that chondrocyte sheet generated by high-density culture of chondrocytes in vitro could cearte a chondrogenic niche in subcutaneous environment and efficiently retain the chondrogenic phenotype of in vitro BMSC engineered cartilage (vitro-BEC). Furthermore, cell tracing results revealed that the regenerated cartilage mainly derived from the implanted vitro-BEC. The current study not only proposes a novel research model for microenvironment simulation but also provides a useful strategy for stable ectopic cartilage regeneration of stem cells. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Epithelial phenotype and the RPE: is the answer blowing in the Wnt?

PubMed

Burke, Janice M

2008-11-01

Cells of the human retinal pigment epithelium (RPE) have a regular epithelial cell shape within the tissue in situ, but for reasons that remain elusive the RPE shows an incomplete and variable ability to re-develop an epithelial phenotype after propagation in vitro. In other epithelial cell cultures, formation of an adherens junction (AJ) composed of E-cadherin plays an important early inductive role in epithelial morphogenesis, but E-cadherin is largely absent from the RPE. In this review, the contribution of cadherins, both minor (E-cadherin) and major (N-cadherin), to RPE phenotype development is discussed. Emphasis is placed on the importance for future studies of actin cytoskeletal remodeling during assembly of the AJ, which in epithelial cells results in an actin organization that is characteristically zonular. Other markers of RPE phenotype that are used to gauge the maturation state of RPE cultures including tissue-specific protein expression, protein polarity, and pigmentation are described. An argument is made that RPE epithelial phenotype, cadherin-based cell-cell adhesion and melanization are linked by a common signaling pathway: the Wnt/beta-catenin pathway. Analyzing this pathway and its intersecting signaling networks is suggested as a useful framework for dissecting the steps in RPE morphogenesis. Also discussed is the effect of aging on RPE phenotype. Preliminary evidence is provided to suggest that light-induced sub-lethal oxidative stress to cultured ARPE-19 cells impairs organelle motility. Organelle translocation, which is mediated by stress-susceptible cytoskeletal scaffolds, is an essential process in cell phenotype development and retention. The observation of impaired organelle motility therefore raises the possibility that low levels of stress, which are believed to accompany RPE aging, may produce subtle disruptions of cell phenotype. Over time these would be expected to diminish the support functions performed by the RPE on behalf of photoreceptors, theoretically contributing to aging retinal disease such as age-related macular degeneration (AMD). Analyzing sub-lethal stress that produces declines in RPE functional efficiency rather than overt cell death is suggested as a useful future direction for understanding the effects of age on RPE organization and physiology. As for phenotype and pigmentation, a role for the Wnt/beta-catenin pathway is also suggested in regulating the RPE response to oxidative stress. Exploration of this pathway in the RPE therefore may provide a unifying strategy for advancing our understanding of both RPE phenotype and the consequences of mild oxidative stress on RPE structure and function.

Single Cell Multi-Omics Technology: Methodology and Application.

PubMed

Hu, Youjin; An, Qin; Sheu, Katherine; Trejo, Brandon; Fan, Shuxin; Guo, Ying

2018-01-01

In the era of precision medicine, multi-omics approaches enable the integration of data from diverse omics platforms, providing multi-faceted insight into the interrelation of these omics layers on disease processes. Single cell sequencing technology can dissect the genotypic and phenotypic heterogeneity of bulk tissue and promises to deepen our understanding of the underlying mechanisms governing both health and disease. Through modification and combination of single cell assays available for transcriptome, genome, epigenome, and proteome profiling, single cell multi-omics approaches have been developed to simultaneously and comprehensively study not only the unique genotypic and phenotypic characteristics of single cells, but also the combined regulatory mechanisms evident only at single cell resolution. In this review, we summarize the state-of-the-art single cell multi-omics methods and discuss their applications, challenges, and future directions.

Single Cell Multi-Omics Technology: Methodology and Application

PubMed Central

Hu, Youjin; An, Qin; Sheu, Katherine; Trejo, Brandon; Fan, Shuxin; Guo, Ying

2018-01-01

In the era of precision medicine, multi-omics approaches enable the integration of data from diverse omics platforms, providing multi-faceted insight into the interrelation of these omics layers on disease processes. Single cell sequencing technology can dissect the genotypic and phenotypic heterogeneity of bulk tissue and promises to deepen our understanding of the underlying mechanisms governing both health and disease. Through modification and combination of single cell assays available for transcriptome, genome, epigenome, and proteome profiling, single cell multi-omics approaches have been developed to simultaneously and comprehensively study not only the unique genotypic and phenotypic characteristics of single cells, but also the combined regulatory mechanisms evident only at single cell resolution. In this review, we summarize the state-of-the-art single cell multi-omics methods and discuss their applications, challenges, and future directions. PMID:29732369

Target identification by image analysis.

PubMed

Fetz, V; Prochnow, H; Brönstrup, M; Sasse, F

2016-05-04

Covering: 1997 to the end of 2015Each biologically active compound induces phenotypic changes in target cells that are characteristic for its mode of action. These phenotypic alterations can be directly observed under the microscope or made visible by labelling structural elements or selected proteins of the cells with dyes. A comparison of the cellular phenotype induced by a compound of interest with the phenotypes of reference compounds with known cellular targets allows predicting its mode of action. While this approach has been successfully applied to the characterization of natural products based on a visual inspection of images, recent studies used automated microscopy and analysis software to increase speed and to reduce subjective interpretation. In this review, we give a general outline of the workflow for manual and automated image analysis, and we highlight natural products whose bacterial and eucaryotic targets could be identified through such approaches.

Phenotype classification of single cells using SRS microscopy, RNA sequencing, and microfluidics (Conference Presentation)

NASA Astrophysics Data System (ADS)

Streets, Aaron M.; Cao, Chen; Zhang, Xiannian; Huang, Yanyi

2016-03-01

Phenotype classification of single cells reveals biological variation that is masked in ensemble measurement. This heterogeneity is found in gene and protein expression as well as in cell morphology. Many techniques are available to probe phenotypic heterogeneity at the single cell level, for example quantitative imaging and single-cell RNA sequencing, but it is difficult to perform multiple assays on the same single cell. In order to directly track correlation between morphology and gene expression at the single cell level, we developed a microfluidic platform for quantitative coherent Raman imaging and immediate RNA sequencing (RNA-Seq) of single cells. With this device we actively sort and trap cells for analysis with stimulated Raman scattering microscopy (SRS). The cells are then processed in parallel pipelines for lysis, and preparation of cDNA for high-throughput transcriptome sequencing. SRS microscopy offers three-dimensional imaging with chemical specificity for quantitative analysis of protein and lipid distribution in single cells. Meanwhile, the microfluidic platform facilitates single-cell manipulation, minimizes contamination, and furthermore, provides improved RNA-Seq detection sensitivity and measurement precision, which is necessary for differentiating biological variability from technical noise. By combining coherent Raman microscopy with RNA sequencing, we can better understand the relationship between cellular morphology and gene expression at the single-cell level.

Receptor control in mesenchymal stem cell engineering

NASA Astrophysics Data System (ADS)

Dalby, Matthew J.; García, Andrés J.; Salmeron-Sanchez, Manuel

2018-03-01

Materials science offers a powerful tool to control mesenchymal stem cell (MSC) growth and differentiation into functional phenotypes. A complex interplay between the extracellular matrix and growth factors guides MSC phenotypes in vivo. In this Review, we discuss materials-based bioengineering approaches to direct MSC fate in vitro and in vivo, mimicking cell-matrix-growth factor crosstalk. We first scrutinize MSC-matrix interactions and how the properties of a material can be tailored to support MSC growth and differentiation in vitro, with an emphasis on MSC self-renewal mechanisms. We then highlight important growth factor signalling pathways and investigate various materials-based strategies for growth factor presentation and delivery. Integrin-growth factor crosstalk in the context of MSC engineering is introduced, and bioinspired material designs with the potential to control the MSC niche phenotype are considered. Finally, we summarize important milestones on the road to MSC engineering for regenerative medicine.

Immune mechanisms in polymyositis and dermatomyositis and potential targets for therapy.

PubMed

Venalis, Paulius; Lundberg, Ingrid E

2014-03-01

PM and DM are characterized clinically by weakness and low endurance of skeletal muscle. Other organs are frequently involved, suggesting that idiopathic inflammatory myopathies (IIMs) are systemic inflammatory diseases. Involvement of immune mechanisms in IIMs is supported by the presence of T cells, macrophages and dendritic cells in muscle tissue, by the presence of autoantibodies and by HLA-DR being a strong genetic risk factor. T cells may have direct and indirect toxic effects on muscle fibres, causing muscle fibre necrosis and muscle weakness, but the target of the immune reaction is not known. A newly identified T cell subset, CD28(null) T cells, may have cytotoxic effects in the CD4(+) and CD8(+) T cell phenotype. These cells are apoptosis resistant and may contribute to treatment resistance. Several myositis-specific autoantibodies have been identified, but they are all directed against ubiquitously expressed autoantigens and the specificity of the T cell reactivity is not known. These autoantibodies are associated with distinct clinical phenotypes and some with distinct molecular pathways; e.g. sera from patients with anti-Jo-1 autoantibodies may activate the type I IFN system and these sera also contain high levels of B cell activating factor compared with other IIM subsets. The characterization of patients into subgroups based on autoantibody profiles seems to be a promising way to learn more about the specificities of the immune reactions. Careful phenotyping of infiltrating immune cells in muscle tissue before and after specific therapies and relating the molecular findings to clinical outcome measures may be another way to improve knowledge on specific immune mechanism in IIMs. Such information will be important for the development of new therapies.

Pax8 modulates the expression of Wnt4 that is necessary for the maintenance of the epithelial phenotype of thyroid cells

PubMed Central

2014-01-01

Background The transcription factor Pax8 is expressed during thyroid development and is involved in the morphogenesis of the thyroid gland and maintenance of the differentiated phenotype. In particular, Pax8 has been shown to regulate genes that are considered markers of thyroid differentiation. Recently, the analysis of the gene expression profile of FRTL-5 differentiated thyroid cells after the silencing of Pax8 identified Wnt4 as a novel target. Like the other members of the Wnt family, Wnt4 has been implicated in several developmental processes including regulation of cell fate and patterning during embryogenesis. To date, the only evidence on Wnt4 in thyroid concerns its down-regulation necessary for the progression of thyroid epithelial tumors. Results Here we demonstrate that Pax8 is involved in the transcriptional modulation of Wnt4 gene expression directly binding to its 5’-flanking region, and that Wnt4 expression in FRTL-5 cells is TSH-dependent. Interestingly, we also show that in thyroid cells a reduced expression of Wnt4 correlates with the alteration of the epithelial phenotype and that the overexpression of Wnt4 in thyroid cancer cells is able to inhibit cellular migration. Conclusions We have identified and characterized a functional Pax8 binding site in the 5’-flanking region of the Wnt4 gene and we show that Pax8 modulates the expression of Wnt4 in thyroid cells. Taken together, our results suggest that in thyroid cells Wnt4 expression correlates with the integrity of the epithelial phenotype and is reduced when this integrity is perturbed. In the end, we would like to suggest that the overexpression of Wnt4 in thyroid cancer cells is able to revert the mesenchymal phenotype. PMID:25270402

Multiple Natural and Experimental Inflammatory Rabbit Lacrimal Gland Phenotypes

PubMed Central

Mircheff, Austin K.; Wang, Yanru; Schechter, Joel E.; Li, Meng; Tong, Warren; Attar, Mayssa; Chengalvala, Murty; Harmuth, Joe; Prusakiewicz, Jeffery J.

2016-01-01

Purpose To investigate lacrimal gland (LG) immunophysiological and immune-mediated inflammatory process (IMIP) phenotype diversity. Methods Ex vivo matured dendritic cells (mDC) were loaded with acinar cell microparticles (MP). Peripheral blood lymphocytes (PBL) were activated in mixed cell reactions with mDC and injected directly into autologous, unilateral LG (1° ATD-LG) of two rabbit cohorts, one naïve, one immunized with a LG lysate membrane fraction (Pi). Autoimmune IgG titers were assayed by ELISA, MCR PBL stimulation indices (SI) by [3H]-thymidine incorporation. Schirmer tests without and with topical anesthetic (STT-I, STT-IA) and rose Bengal (RB) staining tests were performed. H&E and immunohistochemically stained sections were examined. RNA yields and selected transcript abundances were measured. Immune cell number and transcript abundance data were submitted to Principal Component Analysis (PCA). Results Immunizing Pi dose influenced SI but not IgG titers. STT scores were decreased, and rose Bengal scores increased, by day 118 after immunization. Previous immunization exacerbated scores in 1° ATD-eyes and exacerbated 1° ATD-LG atrophy. IMIP were evident in 2° ATD-LG as well as 1° ATD-LG. PCA described diverse immunophysiological phenotypes in control LG and diverse IMIP phenotypes in ATD-LG. IgG titers and SI pre-adoptive transfer were significantly associated with certain post-adoptive transfer IMIP phenotype features, and certain LG IMIP features were significantly associated with RB and STT IA scores. Conclusions The underlying variability of normal states may contribute to the diversity of experimental IMIP phenotypes. The ability to generate and characterize diverse phenotypes may lead to phenotype-specific diagnostic and therapeutic paradigms. PMID:27423911

Maternal allergic disease does not affect the phenotype of T and B cells or the immune response to allergens in neonates.

PubMed

Rindsjö, E; Joerink, M; Johansson, C; Bremme, K; Malmström, V; Scheynius, A

2010-07-01

It is hypothesized that the in utero environment in allergic mothers can affect the neonatal immune responses. The aim of this study was to analyse the effect of maternal allergic disease on cord blood mononuclear cell (CBMC) phenotype and proliferative responses upon allergen stimulation. Peripheral blood mononuclear cells (PBMC) from 12 allergic and 14 nonallergic mothers and CBMC from their children were analysed. In the mothers, we determined cell proliferation, production of IL-4 and expression of FOXP3 in response to allergen stimulation. In the children, we evaluated cell proliferation and FOXP3 expression following allergen stimulation. Furthermore, expression of different homing markers on T cells and regulatory T cells and maturity of the T cells and B cell subsets were evaluated directly ex vivo. The timothy- and birch-allergic mothers responded with increased proliferation and/or IL-4 production towards timothy and birch extract, respectively, when compared to nonallergic mothers. This could not be explained by impairment of FOXP3(+) regulatory T cells in the allergic mothers. CBMC proliferation and FOXP3 expression in response to allergens were not affected by the allergic status of the mother. Also, phenotype of T cells, FOXP3(+) regulatory T cells and B cells was not affected by the allergic status of the mother. Our results suggest that maternal allergic disease has no effect on the neonatal response to allergens or the phenotype of neonatal lymphocytes. The factors studied here could, however, still affect later development of allergy.

Human mesenchymal stem cells cultured on silk hydrogels with variable stiffness and growth factor differentiate into mature smooth muscle cell phenotype.

PubMed

Floren, Michael; Bonani, Walter; Dharmarajan, Anirudh; Motta, Antonella; Migliaresi, Claudio; Tan, Wei

2016-02-01

Cell-matrix and cell-biomolecule interactions play critical roles in a diversity of biological events including cell adhesion, growth, differentiation, and apoptosis. Evidence suggests that a concise crosstalk of these environmental factors may be required to direct stem cell differentiation toward matured cell type and function. However, the culmination of these complex interactions to direct stem cells into highly specific phenotypes in vitro is still widely unknown, particularly in the context of implantable biomaterials. In this study, we utilized tunable hydrogels based on a simple high pressure CO2 method and silk fibroin (SF) the structural protein of Bombyx mori silk fibers. Modification of SF protein starting water solution concentration results in hydrogels of variable stiffness while retaining key structural parameters such as matrix pore size and β-sheet crystallinity. To further resolve the complex crosstalk of chemical signals with matrix properties, we chose to investigate the role of 3D hydrogel stiffness and transforming growth factor (TGF-β1), with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells. Our data revealed the potential to upregulate matured vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72h). Overall, our observations suggest that chemical and physical stimuli within the cellular microenvironment are tightly coupled systems involved in the fate decisions of hMSCs. The production of tunable scaffold materials that are biocompatible and further specialized to mimic tissue-specific niche environments will be of considerable value to future tissue engineering platforms. This article investigates the role of silk fibroin hydrogel stiffness and transforming growth factor (TGF-β1), with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells. Specifically, we demonstrate the upregulation of mature vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72h). Moreover, we demonstrate the potential to direct specialized hMSC differentiation by modulating stiffness and growth factor using silk fibroin, a well-tolerated and -defined biomaterial with an impressive portfolio of tissue engineering applications. Altogether, our study reinforce the fact that complex differentiation protocols may be simplified by engineering the cellular microenvironment on multiple scales, i.e. matrix stiffness with growth factor. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Multiple organ gigantism caused by mutation in VmPPD gene in blackgram (Vigna mungo).

PubMed

Naito, Ken; Takahashi, Yu; Chaitieng, Bubpa; Hirano, Kumi; Kaga, Akito; Takagi, Kyoko; Ogiso-Tanaka, Eri; Thavarasook, Charaspon; Ishimoto, Masao; Tomooka, Norihiko

2017-03-01

Seed size is one of the most important traits in leguminous crops. We obtained a recessive mutant of blackgram that had greatly enlarged leaves, stems and seeds. The mutant produced 100% bigger leaves, 50% more biomass and 70% larger seeds though it produced 40% less number of seeds. We designated the mutant as multiple-organ-gigantism ( mog ) and found the mog phenotype was due to increase in cell numbers but not in cell size. We also found the mog mutant showed a rippled leaf ( rl ) phenotype, which was probably caused by a pleiotropic effect of the mutation. We performed a map-based cloning and successfully identified an 8 bp deletion in the coding sequence of VmPPD gene, an orthologue of Arabidopsis PEAPOD ( PPD ) that regulates arrest of cell divisions in meristematic cells . We found no other mutations in the neighboring genes between the mutant and the wild type. We also knocked down GmPPD genes and reproduced both the mog and rl phenotypes in soybean. Controlling PPD genes to produce the mog phenotype is highly valuable for breeding since larger seed size could directly increase the commercial values of grain legumes.

Multiple organ gigantism caused by mutation in VmPPD gene in blackgram (Vigna mungo)

PubMed Central

Naito, Ken; Takahashi, Yu; Chaitieng, Bubpa; Hirano, Kumi; Kaga, Akito; Takagi, Kyoko; Ogiso-Tanaka, Eri; Thavarasook, Charaspon; Ishimoto, Masao; Tomooka, Norihiko

2017-01-01

Seed size is one of the most important traits in leguminous crops. We obtained a recessive mutant of blackgram that had greatly enlarged leaves, stems and seeds. The mutant produced 100% bigger leaves, 50% more biomass and 70% larger seeds though it produced 40% less number of seeds. We designated the mutant as multiple-organ-gigantism (mog) and found the mog phenotype was due to increase in cell numbers but not in cell size. We also found the mog mutant showed a rippled leaf (rl) phenotype, which was probably caused by a pleiotropic effect of the mutation. We performed a map-based cloning and successfully identified an 8 bp deletion in the coding sequence of VmPPD gene, an orthologue of Arabidopsis PEAPOD (PPD) that regulates arrest of cell divisions in meristematic cells. We found no other mutations in the neighboring genes between the mutant and the wild type. We also knocked down GmPPD genes and reproduced both the mog and rl phenotypes in soybean. Controlling PPD genes to produce the mog phenotype is highly valuable for breeding since larger seed size could directly increase the commercial values of grain legumes. PMID:28588392

A Special Population of Regulatory T Cells Potentiates Muscle Repair

PubMed Central

Burzyn, Dalia; Kuswanto, Wilson; Kolodin, Dmitriy; Shadrach, Jennifer L.; Cerletti, Massimiliano; Jang, Young; Sefik, Esen; Tan, Tze Guan; Wagers, Amy J.; Benoist, Christophe; Mathis, Diane

2014-01-01

SUMMARY Long recognized to be potent suppressors of immune responses, Foxp3+CD4+ regulatory T (Treg) cells are being rediscovered as regulators of nonimmunological processes. We describe a phenotypically and functionally distinct population of Treg cells that rapidly accumulated in the acutely injured skeletal muscle of mice, just as invading myeloidlineage cells switched from a proinflammatory to a proregenerative state. A Treg population of similar phenotype accumulated in muscles of genetically dystrophic mice. Punctual depletion of Treg cells during the repair process prolonged the proinflammatory infiltrate and impaired muscle repair, while treatments that increased or decreased Treg activities diminished or enhanced (respectively) muscle damage in a dystrophy model. Muscle Treg cells expressed the growth factor Amphiregulin, which acted directly on muscle satellite cells in vitro and improved muscle repair in vivo. Thus, Treg cells and their products may provide new therapeutic opportunities for wound repair and muscular dystrophies. PMID:24315098

Epithelial phenotype and the RPE: Is the answer blowing in the Wnt?

PubMed Central

Burke, Janice M.

2008-01-01

Cells of the human retinal pigment epithelium (RPE) have a regular epithelial cell shape within the tissue in situ, but for reasons that remain elusive the RPE shows an incomplete and variable ability to re-develop an epithelial phenotype after propagation in vitro. In other epithelial cell cultures, formation of an adherens junction (AJ) composed of E-cadherin plays an important early inductive role in epithelial morphogenesis, but E-cadherin is largely absent from the RPE. In this review, the contribution of cadherins, both minor (E-cadherin) and major (N-cadherin), to RPE phenotype development is discussed. Emphasis is placed on the importance for future studies of actin cytoskeletal remodeling during assembly of the AJ, which in epithelial cells results in an actin organization that is characteristically zonular. Other markers of RPE phenotype that are used to gauge the maturation state of RPE cultures including tissue-specific protein expression, protein polarity, and pigmentation are described. An argument is made that RPE epithelial phenotype, cadherin-based cell–cell adhesion and melanization are linked by a common signaling pathway: the Wnt/β-catenin pathway. Analyzing this pathway and its intersecting signaling networks is suggested as a useful framework for dissecting the steps in RPE morphogenesis. Also discussed is the effect of aging on RPE phenotype. Preliminary evidence is provided to suggest that light-induced sub-lethal oxidative stress to cultured ARPE-19 cells impairs organelle motility. Organelle translocation, which is mediated by stress-susceptible cytoskeletal scaffolds, is an essential process in cell phenotype development and retention. The observation of impaired organelle motility therefore raises the possibility that low levels of stress, which are believed to accompany RPE aging, may produce subtle disruptions of cell phenotype. Over time these would be expected to diminish the support functions performed by the RPE on behalf of photoreceptors, theoretically contributing to aging retinal disease such as age-related macular degeneration (AMD). Analyzing sub-lethal stress that produces declines in RPE functional efficiency rather than overt cell death is suggested as a useful future direction for understanding the effects of age on RPE organization and physiology. As for phenotype and pigmentation, a role for the Wnt/β-catenin pathway is also suggested in regulating the RPE response to oxidative stress. Exploration of this pathway in the RPE therefore may provide a unifying strategy for advancing our understanding of both RPE phenotype and the consequences of mild oxidative stress on RPE structure and function. PMID:18775790

Consensus-phenotype integration of transcriptomic and metabolomic data implies a role for metabolism in the chemosensitivity of tumour cells.

PubMed

Cavill, Rachel; Kamburov, Atanas; Ellis, James K; Athersuch, Toby J; Blagrove, Marcus S C; Herwig, Ralf; Ebbels, Timothy M D; Keun, Hector C

2011-03-01

Using transcriptomic and metabolomic measurements from the NCI60 cell line panel, together with a novel approach to integration of molecular profile data, we show that the biochemical pathways associated with tumour cell chemosensitivity to platinum-based drugs are highly coincident, i.e. they describe a consensus phenotype. Direct integration of metabolome and transcriptome data at the point of pathway analysis improved the detection of consensus pathways by 76%, and revealed associations between platinum sensitivity and several metabolic pathways that were not visible from transcriptome analysis alone. These pathways included the TCA cycle and pyruvate metabolism, lipoprotein uptake and nucleotide synthesis by both salvage and de novo pathways. Extending the approach across a wide panel of chemotherapeutics, we confirmed the specificity of the metabolic pathway associations to platinum sensitivity. We conclude that metabolic phenotyping could play a role in predicting response to platinum chemotherapy and that consensus-phenotype integration of molecular profiling data is a powerful and versatile tool for both biomarker discovery and for exploring the complex relationships between biological pathways and drug response.

Structure and Expression of Hybrid Dysgenesis-Induced Alleles of the Ovarian Tumor (Otu) Gene in Drosophila Melanogaster

PubMed Central

Sass, G. L.; Mohler, J. D.; Walsh, R. C.; Kalfayan, L. J.; Searles, L. L.

1993-01-01

Mutations at the ovarian tumor (otu) gene of Drosophila melanogaster cause female sterility and generate a range of ovarian phenotypes. Quiescent (QUI) mutants exhibit reduced germ cell proliferation; in oncogenic (ONC) mutants germ cells undergo uncontrolled proliferation generating excessive numbers of undifferentiated cells; the egg chambers of differentiated (DIF) mutants differentiate to variable degrees but fail to complete oogenesis. We have examined mutations caused by insertion and deletion of P elements at the otu gene. The P element insertion sites are upstream of the major otu transcription start sites. In deletion derivatives, the P element, regulatory regions and/or protein coding sequences have been removed. In both insertion and deletion mutants, the level of otu expression correlates directly with the severity of the phenotype: the absence of otu function produces the most severe QUI phenotype while the ONC mutants express lower levels of otu than those which are DIF. The results of this study demonstrate that the diverse mutant phenotypes of otu are the consequence of different levels of otu function. PMID:8436274

P. gingivalis Peptidyl Arginine Deiminase Can Modulate Neutrophil Activity via Infection of Human Dental Stem Cells.

PubMed

Kriebel, Katja; Hieke, Cathleen; Engelmann, Robby; Potempa, Jan; Müller-Hilke, Brigitte; Lang, Hermann; Kreikemeyer, Bernd

2018-06-01

Periodontitis (PD) is a widespread chronic inflammatory disease in the human population. Porphyromonas gingivalis is associated with PD and can citrullinate host proteins via P. gingivalis peptidyl arginine deiminase (PPAD). Here, we hypothesized that infection of human dental follicle stem cells (hDFSCs) with P. gingivalis and subsequent interaction with neutrophils will alter the neutrophil phenotype. To test this hypothesis, we established and analyzed a triple-culture system of neutrophils and hDFSCs primed with P. gingivalis. Mitogen-activated pathway blocking reagents were applied to gain insight into stem cell signaling after infection. Naïve hDFSCs do not influence the neutrophil phenotype. However, infection of hDFSCs with P. gingivalis prolongs the survival of neutrophils and increases their migration. These phenotypic changes depend on direct cellular contacts and PPAD expression by P. gingivalis. Active JNK and ERK pathways in primed hDFSCs are essential for the phenotypic changes in neutrophils. Collectively, our results confirm that P. gingivalis modifies hDFSCs, thereby causing an immune imbalance. © 2018 S. Karger AG, Basel.

Isolation, characterization, and molecular regulation of muscle stem cells

PubMed Central

Fukada, So-ichiro; Ma, Yuran; Ohtani, Takuji; Watanabe, Yoko; Murakami, Satoshi; Yamaguchi, Masahiko

2013-01-01

Skeletal muscle has great regenerative capacity which is dependent on muscle stem cells, also known as satellite cells. A loss of satellite cells and/or their function impairs skeletal muscle regeneration and leads to a loss of skeletal muscle power; therefore, the molecular mechanisms for maintaining satellite cells in a quiescent and undifferentiated state are of great interest in skeletal muscle biology. Many studies have demonstrated proteins expressed by satellite cells, including Pax7, M-cadherin, Cxcr4, syndecan3/4, and c-met. To further characterize satellite cells, we established a method to directly isolate satellite cells using a monoclonal antibody, SM/C-2.6. Using SM/C-2.6 and microarrays, we measured the genes expressed in quiescent satellite cells and demonstrated that Hesr3 may complement Hesr1 in generating quiescent satellite cells. Although Hesr1- or Hesr3-single knockout mice show a normal skeletal muscle phenotype, including satellite cells, Hesr1/Hesr3-double knockout mice show a gradual decrease in the number of satellite cells and increase in regenerative defects dependent on satellite cell numbers. We also observed that a mouse's genetic background affects the regenerative capacity of its skeletal muscle and have established a line of DBA/2-background mdx mice that has a much more severe phenotype than the frequently used C57BL/10-mdx mice. The phenotype of DBA/2-mdx mice also seems to depend on the function of satellite cells. In this review, we summarize the methodology of direct isolation, characterization, and molecular regulation of satellite cells based on our results. The relationship between the regenerative capacity of satellite cells and progression of muscular disorders is also summarized. In the last part, we discuss application of the accumulating scientific information on satellite cells to treatment of patients with muscular disorders. PMID:24273513

Hybrid multiscale modeling and prediction of cancer cell behavior

PubMed Central

Habibi, Jafar

2017-01-01

Background Understanding cancer development crossing several spatial-temporal scales is of great practical significance to better understand and treat cancers. It is difficult to tackle this challenge with pure biological means. Moreover, hybrid modeling techniques have been proposed that combine the advantages of the continuum and the discrete methods to model multiscale problems. Methods In light of these problems, we have proposed a new hybrid vascular model to facilitate the multiscale modeling and simulation of cancer development with respect to the agent-based, cellular automata and machine learning methods. The purpose of this simulation is to create a dataset that can be used for prediction of cell phenotypes. By using a proposed Q-learning based on SVR-NSGA-II method, the cells have the capability to predict their phenotypes autonomously that is, to act on its own without external direction in response to situations it encounters. Results Computational simulations of the model were performed in order to analyze its performance. The most striking feature of our results is that each cell can select its phenotype at each time step according to its condition. We provide evidence that the prediction of cell phenotypes is reliable. Conclusion Our proposed model, which we term a hybrid multiscale modeling of cancer cell behavior, has the potential to combine the best features of both continuum and discrete models. The in silico results indicate that the 3D model can represent key features of cancer growth, angiogenesis, and its related micro-environment and show that the findings are in good agreement with biological tumor behavior. To the best of our knowledge, this paper is the first hybrid vascular multiscale modeling of cancer cell behavior that has the capability to predict cell phenotypes individually by a self-generated dataset. PMID:28846712

Hybrid multiscale modeling and prediction of cancer cell behavior.

PubMed

Zangooei, Mohammad Hossein; Habibi, Jafar

2017-01-01

Understanding cancer development crossing several spatial-temporal scales is of great practical significance to better understand and treat cancers. It is difficult to tackle this challenge with pure biological means. Moreover, hybrid modeling techniques have been proposed that combine the advantages of the continuum and the discrete methods to model multiscale problems. In light of these problems, we have proposed a new hybrid vascular model to facilitate the multiscale modeling and simulation of cancer development with respect to the agent-based, cellular automata and machine learning methods. The purpose of this simulation is to create a dataset that can be used for prediction of cell phenotypes. By using a proposed Q-learning based on SVR-NSGA-II method, the cells have the capability to predict their phenotypes autonomously that is, to act on its own without external direction in response to situations it encounters. Computational simulations of the model were performed in order to analyze its performance. The most striking feature of our results is that each cell can select its phenotype at each time step according to its condition. We provide evidence that the prediction of cell phenotypes is reliable. Our proposed model, which we term a hybrid multiscale modeling of cancer cell behavior, has the potential to combine the best features of both continuum and discrete models. The in silico results indicate that the 3D model can represent key features of cancer growth, angiogenesis, and its related micro-environment and show that the findings are in good agreement with biological tumor behavior. To the best of our knowledge, this paper is the first hybrid vascular multiscale modeling of cancer cell behavior that has the capability to predict cell phenotypes individually by a self-generated dataset.

TFAP2C governs the luminal epithelial phenotype in mammary development and carcinogenesis.

PubMed

Cyr, A R; Kulak, M V; Park, J M; Bogachek, M V; Spanheimer, P M; Woodfield, G W; White-Baer, L S; O'Malley, Y Q; Sugg, S L; Olivier, A K; Zhang, W; Domann, F E; Weigel, R J

2015-01-22

Molecular subtypes of breast cancer are characterized by distinct patterns of gene expression that are predictive of outcome and response to therapy. The luminal breast cancer subtypes are defined by the expression of estrogen receptor-alpha (ERα)-associated genes, many of which are directly responsive to the transcription factor activator protein 2C (TFAP2C). TFAP2C participates in a gene regulatory network controlling cell growth and differentiation during ectodermal development and regulating ESR1/ERα and other luminal cell-associated genes in breast cancer. TFAP2C has been established as a prognostic factor in human breast cancer, however, its role in the establishment and maintenance of the luminal cell phenotype during carcinogenesis and mammary gland development have remained elusive. Herein, we demonstrate a critical role for TFAP2C in maintaining the luminal phenotype in human breast cancer and in influencing the luminal cell phenotype during normal mammary development. Knockdown of TFAP2C in luminal breast carcinoma cells induced epithelial-mesenchymal transition with morphological and phenotypic changes characterized by a loss of luminal-associated gene expression and a concomitant gain of basal-associated gene expression. Conditional knockout of the mouse homolog of TFAP2C, Tcfap2c, in mouse mammary epithelium driven by MMTV-Cre promoted aberrant growth of the mammary tree leading to a reduction in the CD24(hi)/CD49f(mid) luminal cell population and concomitant gain of the CD24(mid)/CD49f(hi) basal cell population at maturity. Our results establish TFAP2C as a key transcriptional regulator for maintaining the luminal phenotype in human breast carcinoma. Furthermore, Tcfap2c influences development of the luminal cell type during mammary development. The data suggest that TFAP2C has an important role in regulated luminal-specific genes and may be a viable therapeutic target in breast cancer.

Asymptomatic memory CD8+ T cells

PubMed Central

Khan, Arif Azam; Srivastava, Ruchi; Lopes, Patricia Prado; Wang, Christine; Pham, Thanh T; Cochrane, Justin; Thai, Nhi Thi Uyen; Gutierrez, Lucas; BenMohamed, Lbachir

2014-01-01

Generation and maintenance of high quantity and quality memory CD8+ T cells determine the level of protection from viral, bacterial, and parasitic re-infections, and hence constitutes a primary goal for T cell epitope-based human vaccines and immunotherapeutics. Phenotypically and functionally characterizing memory CD8+ T cells that provide protection against herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) infections, which cause blinding ocular herpes, genital herpes, and oro-facial herpes, is critical for better vaccine design. We have recently categorized 2 new major sub-populations of memory symptomatic and asymptomatic CD8+ T cells based on their phenotype, protective vs. pathogenic function, and anatomical locations. In this report we are discussing a new direction in developing T cell-based human herpes vaccines and immunotherapeutics based on the emerging new concept of “symptomatic and asymptomatic memory CD8+ T cells.” PMID:24499824

Two subpopulations of Crocosphaera watsonii have distinct distributions in the North and South Pacific.

PubMed

Bench, Shellie R; Frank, Ildiko; Robidart, Julie; Zehr, Jonathan P

2016-02-01

Crocosphaera watsonii is a unicellular nitrogen (N2)-fixing cyanobacterium with ecological importance in oligotrophic oceans. In cultivated strains there are two phenotypes of C. watsonii (large and small cells) with differences that could differentially impact biogeochemical processes. Recent work has shown the phenotypes diverged through loss or addition of type-specific genes in a fraction of their genomes, whereas the rest of the genomes were maintained at 99-100% DNA identity. Previous molecular assays for C. watsonii abundances targeted the conserved regions and therefore could not differentiate between phenotypes, so their relative distributions in natural communities were unknown. To determine phenotype distributions, this study developed and applied type-specific quantitative polymerase chain reaction assays to samples from the North and South Pacific. Abundances of both Crocosphaera types declined sharply with depth between 45 and 75 m in both sites. In surface water small cells were 10-100 times more abundant than large cells in the N. Pacific, whereas in the S. Pacific the two phenotypes were nearly equal. Evidence for large cell aggregation was only found in N. Pacific samples. The differences in C. watsonii sub-populations in the North and South Pacific have direct implications for biogeochemistry and carbon export in oligotrophic gyres. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Autoreactive T cells from patients with myasthenia gravis are characterized by elevated IL-17, IFN-γ and GM-CSF and diminished IL-10 production

PubMed Central

Cao, Yonghao; Amezquita, Robert A.; Kleinstein, Steven H.; Stathopoulos, Panos; Nowak, Richard J.; O’Connor, Kevin C.

2016-01-01

Myasthenia gravis (MG) is a prototypical autoimmune disease that is among the few for which the target antigen and the pathogenic autoantibodies are clearly defined. The pathology of the disease is affected by autoantibodies directed toward the acetylcholine receptor (AChR). Mature, antigen-experienced B cells rely on the action of helper T cells to produce these pathogenic antibodies. The phenotype of the MG antigen-reactive T cell compartment is not well defined, thus we sought to determine whether such cells exhibit both a pro-inflammatory and pathogenic phenotype. A novel T cell library assay that affords multi-parameter interrogation of rare antigen-reactive CD4+ T cells was applied. Proliferation and cytokine production in response to both AChR and control antigens were measured from 3,120 T cell libraries derived from eleven MG subjects and paired healthy controls. The frequency of CCR6+ memory T cells from MG subjects proliferating in response to AChR-derived peptides was significantly higher than that of healthy controls. Production of both IFN-γ and IL-17, in response to AChR was also restricted to the CCR6+ memory T cell compartment in the MG cohort indicating a pro-inflammatory phenotype. These T cells also included an elevated expression of GM-CSF and absence of IL-10 expression, indicating a pro-inflammatory and pathogenic phenotype. This component of the autoimmune response in MG is of particular importance when considering the durability of MG treatment strategies that eliminate B cells, as the autoreactive T cells could renew autoimmunity in the reconstituted B cell compartment with ensuing clinical manifestations. PMID:26826242

Long term storage in liquid nitrogen leads to only minor phenotypic and gene expression changes in the mammary carcinoma model cell line BT474.

PubMed

Fazekas, Judit; Grunt, Thomas W; Jensen-Jarolim, Erika; Singer, Josef

2017-05-23

Cancer cell lines are indispensible surrogate models in cancer research, as they can be used off-the-shelf, expanded to the desired extent, easily modified and exchanged between research groups for affirmation, reproduction or follow-up experiments.As malignant cells are prone to genomic instability, phenotypical changes may occur after certain passages in culture. Thus, cell lines have to be regularly authenticated to ensure data quality. In between experiments these cell lines are often stored in liquid nitrogen for extended time periods.Although freezing of cells is a necessary evil, little research is performed on how long-term storage affects cancer cell lines. Therefore, this study investigated the effects of a 28-year long liquid nitrogen storage period on BT474 cells with regard to phenotypical changes, differences in cell-surface receptor expression as well as cytokine and gene expressional variations. Two batches of BT474 cells, one frozen in 1986, the other directly purchased from ATCC were investigated by light microscopy, cell growth analysis, flow cytometry and cytokine as well as whole-transcriptome expression profiling. The cell lines were morphologically indifferent and showed similar growth rates and similar cell-surface receptor expression. Transcriptome analysis revealed significant differences in only 26 of 40,716 investigated RefSeq transcripts with 4 of them being up-regulated and 22 down-regulated. This study demonstrates that even after very long periods of storage in liquid nitrogen, cancer cell lines display only minimal changes in their gene expression profiles. However, also such minor changes should be carefully assessed before continuation of experiments, especially if phenotypic alterations can be additionally observed.

MicroRNA-124 controls human vascular smooth muscle cell phenotypic switch via Sp1.

PubMed

Tang, Yangfeng; Yu, Shangyi; Liu, Yang; Zhang, Jiajun; Han, Lin; Xu, Zhiyun

2017-09-01

Phenotypic switch of vascular smooth muscle cells (VSMCs) plays an important role in the pathogenesis of atherosclerosis and aortic dissection. However, the mechanisms of phenotypic modulation are still unclear. MicroRNAs have emerged as important regulators of VSMC function. We recently found that microRNA-124 (miR-124) was downregulated in proliferative vascular diseases that were characterized by a VSMC phenotypic switch. Therefore, we speculated that the aberrant expression of miR-124 might play a critical role in human aortic VSMC phenotypic switch. Using quantitative RT-PCR, we found that miR-124 was dramatically downregulated in the aortic media of clinical specimens of the dissected aorta and correlated with molecular markers of the contractile VSMC phenotype. Overexpression of miR-124 by mimicking transfection significantly attenuated platelet-derived growth factor-BB-induced human aortic VSMC proliferation and phenotypic switch. Furthermore, we identified specificity protein 1 (Sp1) as the downstream target of miR-124. A luciferase reporter assay was used to confirm direct miR-124 targeting of the 3'-untranslated region of the Sp1 gene and repression of Sp1 expression in human aortic VSMCs. Furthermore, constitutively active Sp1 in miR-124-overexpressing VSMCs reversed the antiproliferative effects of miR-124. These results demonstrated a novel mechanism of miR-124 modulation of VSMC phenotypic switch by targeting Sp1 expression. NEW & NOTEWORTHY Previous studies have demonstrated that miR-124 is involved in the proliferation of a variety of cell types. However, miRNAs are expressed in a tissue-specific manner. We first identified miR-124 as a critical regulator in human aortic vascular smooth muscle cell differentiation, proliferation, and phenotype switch by targeting the 3'-untranslated region of specificity protein 1. Copyright © 2017 the American Physiological Society.

Drug-induced death signaling strategy rapidly predicts cancer response to chemotherapy.

PubMed

Montero, Joan; Sarosiek, Kristopher A; DeAngelo, Joseph D; Maertens, Ophélia; Ryan, Jeremy; Ercan, Dalia; Piao, Huiying; Horowitz, Neil S; Berkowitz, Ross S; Matulonis, Ursula; Jänne, Pasi A; Amrein, Philip C; Cichowski, Karen; Drapkin, Ronny; Letai, Anthony

2015-02-26

There is a lack of effective predictive biomarkers to precisely assign optimal therapy to cancer patients. While most efforts are directed at inferring drug response phenotype based on genotype, there is very focused and useful phenotypic information to be gained from directly perturbing the patient's living cancer cell with the drug(s) in question. To satisfy this unmet need, we developed the Dynamic BH3 Profiling technique to measure early changes in net pro-apoptotic signaling at the mitochondrion ("priming") induced by chemotherapeutic agents in cancer cells, not requiring prolonged ex vivo culture. We find in cell line and clinical experiments that early drug-induced death signaling measured by Dynamic BH3 Profiling predicts chemotherapy response across many cancer types and many agents, including combinations of chemotherapies. We propose that Dynamic BH3 Profiling can be used as a broadly applicable predictive biomarker to predict cytotoxic response of cancers to chemotherapeutics in vivo. Copyright © 2015 Elsevier Inc. All rights reserved.

Modulation of invasive phenotype by interstitial pressure-driven convection in aggregates of human breast cancer cells.

PubMed

Tien, Joe; Truslow, James G; Nelson, Celeste M

2012-01-01

This paper reports the effect of elevated pressure on the invasive phenotype of patterned three-dimensional (3D) aggregates of MDA-MB-231 human breast cancer cells. We found that the directionality of the interstitial pressure profile altered the frequency of invasion by cells located at the surface of an aggregate. In particular, application of pressure at one end of an aggregate suppressed invasion at the opposite end. Experimental alteration of the configuration of cell aggregates and computational modeling of the resulting flow and solute concentration profiles revealed that elevated pressure inhibited invasion by altering the chemical composition of the interstitial fluid near the surface of the aggregate. Our data reveal a link between hydrostatic pressure, interstitial convection, and invasion.

The process behind the expression of mdr-1/P-gp and mrp/MRP in human leukemia/lymphoma.

PubMed

Hirose, Masao

2009-04-01

There is a controversy over the link between phenotypes of multidrug resistance (MDR) and clinical outcome in leukemia/lymphoma patients. This may be because the process behind the induction and loss of expression of genotypes and phenotypes by which MDR develops and the role of MDR in fresh cells of human leukemia/lymphoma are not clearly defined. P-glycoprotein (P-gp) increased and decreased along with mdr-1 expression in three cell lines out of five vincristine (VCR)-resistant cell lines. MRP appeared with increased mrp expression in the other two cell lines. After the drug was removed from the culture system, mdr-1/P-gp changed in parallel with the level of VCR resistance, although mrp and MRP did not. It was concluded that P-gp is directly derived from mdr-1 and that mdr-1/P-gp supports the VCR-resistance but mrp/MRP is not directly linked to the VCR-resistance. These results should contribute to a better understanding of MDR phenomenon in cancer.

Urokinase Receptor Counteracts Vascular Smooth Muscle Cell Functional Changes Induced by Surface Topography

PubMed Central

Kiyan, Yulia; Kurselis, Kestutis; Kiyan, Roman; Haller, Hermann; Chichkov, Boris N.; Dumler, Inna

2013-01-01

Current treatments for human coronary artery disease necessitate the development of the next generations of vascular bioimplants. Recent reports provide evidence that controlling cell orientation and morphology through topographical patterning might be beneficial for bioimplants and tissue engineering scaffolds. However, a concise understanding of cellular events underlying cell-biomaterial interaction remains missing. In this study, applying methods of laser material processing, we aimed to obtain useful markers to guide in the choice of better vascular biomaterials. Our data show that topographically treated human primary vascular smooth muscle cells (VSMC) have a distinct differentiation profile. In particular, cultivation of VSMC on the microgrooved biocompatible polymer E-shell induces VSMC modulation from synthetic to contractile phenotype and directs formation and maintaining of cell-cell communication and adhesion structures. We show that the urokinase receptor (uPAR) interferes with VSMC behavior on microstructured surfaces and serves as a critical regulator of VSMC functional fate. Our findings suggest that microtopography of the E-shell polymer could be important in determining VSMC phenotype and cytoskeleton organization. They further suggest uPAR as a useful target in the development of predictive models for clinical VSMC phenotyping on functional advanced biomaterials. PMID:23843899

The directed differentiation of human iPS cells into kidney podocytes.

PubMed

Song, Bi; Smink, Alexandra M; Jones, Christina V; Callaghan, Judy M; Firth, Stephen D; Bernard, Claude A; Laslett, Andrew L; Kerr, Peter G; Ricardo, Sharon D

2012-01-01

The loss of glomerular podocytes is a key event in the progression of chronic kidney disease resulting in proteinuria and declining function. Podocytes are slow cycling cells that are considered terminally differentiated. Here we provide the first report of the directed differentiation of induced pluripotent stem (iPS) cells to generate kidney cells with podocyte features. The iPS-derived podocytes share a morphological phenotype analogous with cultured human podocytes. Following 10 days of directed differentiation, iPS podocytes had an up-regulated expression of mRNA and protein localization for podocyte markers including synaptopodin, nephrin and Wilm's tumour protein (WT1), combined with a down-regulation of the stem cell marker OCT3/4. In contrast to human podocytes that become quiescent in culture, iPS-derived cells maintain a proliferative capacity suggestive of a more immature phenotype. The transduction of iPS podocytes with fluorescent labeled-talin that were immunostained with podocin showed a cytoplasmic contractile response to angiotensin II (AII). A permeability assay provided functional evidence of albumin uptake in the cytoplasm of iPS podocytes comparable to human podocytes. Moreover, labeled iPS-derived podocytes were found to integrate into reaggregated metanephric kidney explants where they incorporated into developing glomeruli and co-expressed WT1. This study establishes the differentiation of iPS cells to kidney podocytes that will be useful for screening new treatments, understanding podocyte pathogenesis, and offering possibilities for regenerative medicine.

Phenotypic variability and selection of lipid-producing microalgae in a microfluidic centrifuge

NASA Astrophysics Data System (ADS)

Estévez-Torres, André.; Mestler, Troy; Austin, Robert H.

2010-03-01

Isogenic cells are known to display various expression levels that may result in different phenotypes within a population. Here we focus on the phenotypic variability of a species of unicellular algae that produce neutral lipids. Lipid-producing algae are one of the most promising sources of biofuel. We have implemented a simple microfluidic method to assess lipid-production variability in a population of algae that relays on density differences. We will discuss the reasons of this variability and address the promising avenues of this technique for directing the evolution of algae towards high lipid productivity.

Cell-surface central nervous system autoantibodies: Clinical relevance and emerging paradigms

PubMed Central

Irani, Sarosh R; Gelfand, Jeffrey M; Al-Diwani, Adam; Vincent, Angela

2014-01-01

The recent discovery of several potentially pathogenic autoantibodies has helped identify patients with clinically distinctive central nervous system diseases that appear to benefit from immunotherapy. The associated autoantibodies are directed against the extracellular domains of cell-surface–expressed neuronal or glial proteins such as LGI1, N-methyl-D-aspartate receptor, and aquaporin-4. The original descriptions of the associated clinical syndromes were phenotypically well circumscribed. However, as availability of antibody testing has increased, the range of associated patient phenotypes and demographics has expanded. This in turn has led to the recognition of more immunotherapy-responsive syndromes in patients presenting with cognitive and behavioral problems, seizures, movement disorders, psychiatric features, and demyelinating disease. Although antibody detection remains diagnostically important, clinical recognition of these distinctive syndromes should ensure early and appropriate immunotherapy administration. We review the emerging paradigm of cell-surface–directed antibody–mediated neurological diseases, describe how the associated disease spectrums have broadened since the original descriptions, discuss some of the methodological issues regarding techniques for antibody detection and emphasize considerations surrounding immunotherapy administration. As these disorders continue to reach mainstream neurology and even psychiatry, more cell-surface–directed antibodies will be discovered, and their possible relevance to other more common disease presentations should become more clearly defined. PMID:24930434

Neurogenesis and ontogeny of specific cell phenotypes within the hamster suprachiasmatic nucleus.

PubMed

Antle, Michael C; LeSauter, Joseph; Silver, Rae

2005-06-09

The hamster suprachiasmatic nucleus (SCN) is anatomically and functionally heterogeneous. A group of cells in the SCN shell, delineated by vasopressin-ergic neurons, are rhythmic with respect to Period gene expression and electrical activity but do not receive direct retinal input. In contrast, some cells in the SCN core, marked by neurons containing calbindin-D28k, gastrin-releasing peptide (GRP), substance P (SP), and vasoactive intestinal polypeptide (VIP), are not rhythmic with respect to Period gene expression and electrical activity but do receive direct retinal input. Examination of the timing of neurogenesis using bromodeoxyuridine indicates that SCN cells are born between embryonic day 9.5 and 12.5. Calbindin, GRP, substance P, and VIP cells are born only during early SCN neurogenesis, between embryonic days 9.5-11.0. Vasopressin cells are born over the whole period of SCN neurogenesis, appearing as late as embryonic day 12.5. Examination of the ontogeny of peptide expression in these cell types reveals transient expression of calbindin in a cluster of dorsolateral SCN cells on postnatal days 1-2. The adult pattern of calbindin expression is detected in a different ventrolateral cell cluster starting on postnatal day 2. GRP and SP expression appear on postnatal day 8 and 10, respectively, after the retinohypothalamic tract has innervated the SCN. In summary, the present study describes the ontogeny-specific peptidergic phenotypes in the SCN and compares these developmental patterns to previously identified patterns in the appearance of circadian functions. These comparisons suggest the possibility that these coincident appearances may be causally related, with the direction of causation to be determined.

IL-10-overexpressing B cells regulate innate and adaptive immune responses.

PubMed

Stanic, Barbara; van de Veen, Willem; Wirz, Oliver F; Rückert, Beate; Morita, Hideaki; Söllner, Stefan; Akdis, Cezmi A; Akdis, Mübeccel

2015-03-01

Distinct human IL-10-producing B-cell subsets with immunoregulatory properties have been described. However, the broader spectrum of their direct cellular targets and suppressive mechanisms has not been extensively studied, particularly in relation to direct and indirect IL-10-mediated functions. The aim of the study was to investigate the effects of IL-10 overexpression on the phenotype and immunoregulatory capacity of B cells. Primary human B cells were transfected with hIL-10, and IL-10-overexpressing B cells were characterized for cytokine and immunoglobulin production by means of specific ELISA and bead-based assays. Antigen presentation, costimulation capacity, and transcription factor signatures were analyzed by means of flow cytometry and quantitative RT-PCR. Effects of IL-10-overexpresing B cells on Toll-like receptor-triggered cytokine release from PBMCs, LPS-triggered maturation of monocyte-derived dendritic cells, and tetanus toxoid-induced PBMC proliferation were assessed in autologous cocultures. IL-10-overexpressing B cells acquired a prominent immunoregulatory profile comprising upregulation of suppressor of cytokine signaling 3 (SOCS3), glycoprotein A repetitions predominant (GARP), the IL-2 receptor α chain (CD25), and programmed cell death 1 ligand 1 (PD-L1). Concurrently, their secretion profile was characterized by a significant reduction in levels of proinflammatory cytokines (TNF-α, IL-8, and macrophage inflammatory protein 1α) and augmented production of anti-inflammatory IL-1 receptor antagonist and vascular endothelial growth factor. Furthermore, IL-10 overexpression was associated with a decrease in costimulatory potential. IL-10-overexpressing B cells secreted less IgE and potently suppressed proinflammatory cytokines in PBMCs, maturation of monocyte-derived dendritic cells (rendering their profile to regulatory phenotype), and antigen-specific proliferation in vitro. Our data demonstrate an essential role for IL-10 in inducing an immunoregulatory phenotype in B cells that exerts substantial anti-inflammatory and immunosuppressive functions. Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

KRAS Mutation and Epithelial-Macrophage Interplay in Pancreatic Neoplastic Transformation.

PubMed

Bishehsari, Faraz; Zhang, Lijuan; Barlass, Usman; Preite, Nailliw; Turturro, Sanja; Najor, Matthew S; Shetuni, Brandon B; Zayas, Janet P; Mahdavinia, Mahboobeh; Abukhdeir, Abde M; Keshavarzian, Ali

2018-05-14

Pancreatic ductal adenocarcinoma (PDA) is characterized by epithelial mutations in KRAS and prominent tumor-associated inflammation, including macrophage infiltration. But knowledge of early interactions between neoplastic epithelium and macrophages in PDA carcinogenesis is limited. Using a pancreatic organoid model, we found that the expression of mutant KRAS in organoids increased i) ductal to acinar gene expression ratios, ii) epithelial cells proliferation, and iii) colony formation capacity in vitro, and endowed pancreatic cells with the ability to generate neoplastic tumors in vivo. KRAS mutations induced a pro-tumorigenic phenotype in macrophages. Altered macrophages decreased epithelial Pigment Epithelial Derived Factor (PEDF) expression and induced a cancerous phenotype. We validated our findings using annotated patient samples from The Cancer Genome Atlas (TCGA) as well as in our human PDA specimens. Epithelium-macrophage cross talk occurs early in pancreatic carcinogenesis where KRAS directly induces cancer-related phenotypes in epithelium, and also promotes a pro-tumorigenic phenotype in macrophages, in turn augmenting neoplastic growth. This article is protected by copyright. All rights reserved. © 2018 UICC.

ω-3 polyunsaturated fatty acids direct differentiation of the membrane phenotype in mesenchymal stem cells to potentiate osteogenesis

PubMed Central

Levental, Kandice R.; Surma, Michal A.; Skinkle, Allison D.; Lorent, Joseph H.; Zhou, Yong; Klose, Christian; Chang, Jeffrey T.; Hancock, John F.; Levental, Ilya

2017-01-01

Mammalian cells produce hundreds of dynamically regulated lipid species that are actively turned over and trafficked to produce functional membranes. These lipid repertoires are susceptible to perturbations from dietary sources, with potentially profound physiological consequences. However, neither the lipid repertoires of various cellular membranes, their modulation by dietary fats, nor their effects on cellular phenotypes have been widely explored. We report that differentiation of human mesenchymal stem cells (MSCs) into osteoblasts or adipocytes results in extensive remodeling of the plasma membrane (PM), producing cell-specific membrane compositions and biophysical properties. The distinct features of osteoblast PMs enabled rational engineering of membrane phenotypes to modulate differentiation in MSCs. Specifically, supplementation with docosahexaenoic acid (DHA), a lipid component characteristic of osteoblast membranes, induced broad lipidomic remodeling in MSCs that reproduced compositional and structural aspects of the osteoblastic PM phenotype. The PM changes induced by DHA supplementation potentiated osteogenic differentiation of MSCs concurrent with enhanced Akt activation at the PM. These observations prompt a model wherein the DHA-induced lipidome leads to more stable membrane microdomains, which serve to increase Akt activity and thereby enhance osteogenic differentiation. More broadly, our investigations suggest a general mechanism by which dietary fats affect cellular physiology through remodeling of membrane lipidomes, biophysical properties, and signaling. PMID:29134198

A Bovine Lymphosarcoma Cell Line Infected with Theileria annulata Exhibits an Irreversible Reconfiguration of Host Cell Gene Expression

PubMed Central

Durrani, Zeeshan; Pillai, Sreerekha S.; Baird, Margaret; Shiels, Brian R.

2013-01-01

Theileria annulata, an intracellular parasite of bovine lymphoid cells, induces substantial phenotypic alterations to its host cell including continuous proliferation, cytoskeletal changes and resistance to apoptosis. While parasite induced modulation of host cell signal transduction pathways and NFκB activation are established, there remains considerable speculation on the complexities of the parasite directed control mechanisms that govern these radical changes to the host cell. Our objectives in this study were to provide a comprehensive analysis of the global changes to host cell gene expression with emphasis on those that result from direct intervention by the parasite. By using comparative microarray analysis of an uninfected bovine cell line and its Theileria infected counterpart, in conjunction with use of the specific parasitacidal agent, buparvaquone, we have identified a large number of host cell gene expression changes that result from parasite infection. Our results indicate that the viable parasite can irreversibly modify the transformed phenotype of a bovine cell line. Fifty percent of genes with altered expression failed to show a reversible response to parasite death, a possible contributing factor to initiation of host cell apoptosis. The genes that did show an early predicted response to loss of parasite viability highlighted a sub-group of genes that are likely to be under direct control by parasite infection. Network and pathway analysis demonstrated that this sub-group is significantly enriched for genes involved in regulation of chromatin modification and gene expression. The results provide evidence that the Theileria parasite has the regulatory capacity to generate widespread change to host cell gene expression in a complex and largely irreversible manner. PMID:23840536

Donor‐Dependent and Other Nondefined Factors Have Greater Influence on the Hepatic Phenotype Than the Starting Cell Type in Induced Pluripotent Stem Cell Derived Hepatocyte‐Like Cells

PubMed Central

Heslop, James A.; Kia, Richard; Pridgeon, Christopher S.; Sison‐Young, Rowena L.; Liloglou, Triantafillos; Elmasry, Mohamed; Fenwick, Stephen W.; Mills, John S.; Kitteringham, Neil R.; Park, Bong K.

2017-01-01

Abstract Drug‐induced liver injury is the greatest cause of post‐marketing drug withdrawal; therefore, substantial resources are directed toward triaging potentially dangerous new compounds at all stages of drug development. One of the major factors preventing effective screening of new compounds is the lack of a predictive in vitro model of hepatotoxicity. Primary human hepatocytes offer a metabolically relevant model for which the molecular initiating events of hepatotoxicity can be examined; however, these cells vary greatly between donors and dedifferentiate rapidly in culture. Induced pluripotent stem cell (iPSC)‐derived hepatocyte‐like cells (HLCs) offer a reproducible, physiologically relevant and genotypically normal model cell; however, current differentiation protocols produce HLCs with a relatively immature phenotype. During the reprogramming of somatic cells, the epigenome undergoes dramatic changes; however, this “resetting” is a gradual process, resulting in an altered differentiation propensity, skewed toward the lineage of origin, particularly in early passage cultures. We, therefore, performed a comparison of human hepatocyte‐ and dermal fibroblast‐derived iPSCs, assessing the impact of epigenetic memory at all stages of HLC differentiation. These results provide the first isogenic assessment of the starting cell type in human iPSC‐derived HLCs. Despite a trend toward improvement in hepatic phenotype in albumin secretion and gene expression, few significant differences in hepatic differentiation capacity were found between hepatocyte and fibroblast‐derived iPSCs. We conclude that the donor and inter‐clonal differences have a greater influence on the hepatocyte phenotypic maturity than the starting cell type. Therefore, it is not necessary to use human hepatocytes for generating iPSC‐derived HLCs. Stem Cells Translational Medicine 2017;6:1321–1331 PMID:28456008

New Advanced Technologies in Stem Cell Therapy

DTIC Science & Technology

2012-09-01

directions for this project include investigating modulation of the IKK/NF-kB pathway as a means to rejuvenate the phenotype of aged muscle stem and...Reference 1. Conboy IM, Conboy MJ, Wagers AJ, Girma ER, Weissman IL, Rando TA. Rejuvenation of aged progenitor cells by exposure to a young...the influence that age plays on the regeneration capacity of the cells. Study Design: We will investigate the effects of cell survival, proliferation

A new Gsdma3 mutation affecting anagen phase of first hair cycle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tanaka, Shigekazu; Department of Genetics, School of Life Science, Graduate University for Advanced Studies, 1111 Yata, Mishima, Shizuoka 411-8540; Tamura, Masaru

2007-08-10

Recombination-induced mutation 3 (Rim3) is a spontaneous mouse mutation that exhibits dominant phenotype of hyperkeratosis and hair loss. Fine linkage analysis of Rim3 and sequencing revealed a novel single point mutation, G1124A leading to Ala348Thr, in Gsdma3 in chromosome 11. Transgenesis with BAC DNA harboring the Rim3-type Gsdma3 recaptured the Rim3 phenotype, providing direct evidence that Gsdma3 is the causative gene of Rim3. We examined the spatial expression of Gsdma3 and characterized the Rim3 phenotype in detail. Gsdma3 is expressed in differentiated epidermal cells in the skin, but not in the proliferating epidermal cells. Histological analysis of Rim3 mutant showedmore » hyperplasia of the epidermal cells in the upper hair follicles and abnormal anagen phase at the first hair cycle. Furthermore, immunohistochemical analysis revealed hyperproliferation and misdifferentiation of the upper follicular epidermis in Rim3 mutant. These results suggest that Gsdma3 is involved in the proliferation and differentiation of epidermal stem cells.« less

Targeted modification of homogalacturonan by transgenic expression of a fungal polygalacturonase alters plant growth.

PubMed

Capodicasa, Cristina; Vairo, Donatella; Zabotina, Olga; McCartney, Lesley; Caprari, Claudio; Mattei, Benedetta; Manfredini, Cinzia; Aracri, Benedetto; Benen, Jacques; Knox, J Paul; De Lorenzo, Giulia; Cervone, Felice

2004-07-01

Pectins are a highly complex family of cell wall polysaccharides comprised of homogalacturonan (HGA), rhamnogalacturonan I and rhamnogalacturonan II. We have specifically modified HGA in both tobacco (Nicotiana tabacum) and Arabidopsis by expressing the endopolygalacturonase II of Aspergillus niger (AnPGII). Cell walls of transgenic tobacco plants showed a 25% reduction in GalUA content as compared with the wild type and a reduced content of deesterified HGA as detected by antibody labeling. Neutral sugars remained unchanged apart from a slight increase of Rha, Ara, and Gal. Both transgenic tobacco and Arabidopsis were dwarfed, indicating that unesterified HGA is a critical factor for plant cell growth. The dwarf phenotypes were associated with AnPGII activity as demonstrated by the observation that the mutant phenotype of tobacco was completely reverted by crossing the dwarfed plants with plants expressing PGIP2, a strong inhibitor of AnPGII. The mutant phenotype in Arabidopsis did not appear when transformation was performed with a gene encoding AnPGII inactivated by site directed mutagenesis.

Does the central dogma still stand?

PubMed

Koonin, Eugene V

2012-08-23

Prions are agents of analog, protein conformation-based inheritance that can confer beneficial phenotypes to cells, especially under stress. Combined with genetic variation, prion-mediated inheritance can be channeled into prion-independent genomic inheritance. Latest screening shows that prions are common, at least in fungi. Thus, there is non-negligible flow of information from proteins to the genome in modern cells, in a direct violation of the Central Dogma of molecular biology. The prion-mediated heredity that violates the Central Dogma appears to be a specific, most radical manifestation of the widespread assimilation of protein (epigenetic) variation into genetic variation. The epigenetic variation precedes and facilitates genetic adaptation through a general 'look-ahead effect' of phenotypic mutations. This direction of the information flow is likely to be one of the important routes of environment-genome interaction and could substantially contribute to the evolution of complex adaptive traits.

Anatomical, biochemical, and photosynthetic responses to recent allopolyploidy in Glycine dolichocarpa (Fabaceae).

PubMed

Coate, Jeremy E; Luciano, Amelia K; Seralathan, Vasu; Minchew, Kevin J; Owens, Tom G; Doyle, Jeff J

2012-01-01

Previous studies have shown that polyploidy has pronounced effects on photosynthesis. Most of these studies have focused on synthetic or recently formed autopolyploids, and comparatively little is known about the integrated effects of natural allopolyploidy, which involves hybridity and genome doubling and often incorporates multiple genotypes through recurrent origins and lineage recombination. Glycine dolichocarpa (designated T2) is a natural allotetraploid with multiple origins. We quantified 21 anatomical, biochemical, and physiological phenotypes relating to photosynthesis in T2 and its diploid progenitors, G. tomentella (D3) and G. syndetika (D4). To assess how direction of cross affects these phenotypes, we included three T2 accessions having D3-like plastids (T2(D3)) and two accessions having D4-like plastids (T2(D4)). T2 accessions were transgressive (more extreme than any diploid accession) for 17 of 21 phenotypes, and species means differed significantly in T2 vs. both progenitors for four of 21 phenotypes (higher for guard cell length, electron transport capacity [J(max)] per palisade cell, and J(max) per mesophyll cell; lower for palisade cells per unit leaf area). Within T2, four of 21 parameters differed significantly between T2(D3) and T2(D4) (palisade cell volume; chloroplast number and volume per unit leaf area; and J(max) per unit leaf area). T2 is characterized by transgressive photosynthesis-related phenotypes (including an ca. 2-fold increase in J(max) per cell), as well as by significant intraspecies variation correlating with plastid type. These data indicate prominent roles for both nucleotypic effects and cytoplasmic factors in photosynthetic responses to allopolyploidy.

A rare case of anti-jk3 antibody detected on pre-transfusion investigation.

PubMed

Yousuf, Rabeya; Abdul Aziz, Suria; Yusof, Nurasyikin; Leong, Chooi-Fun

2014-09-01

We report a 47-year-old Malay lady, para 4 + 1, with known medical history of hypertension whom presented at Emergency Department with severe anaemia, most likely secondary to menorrhagia caused by uterine fibroids. Her haemoglobin was 5.5 g/dl and she was transfused with three units of packed cell without any adverse reaction, her haemoglobin level increased to 9.8 g/dl. She was then planned for total abdominal hysterectomy and bilateral salpingo-oophorectomy later. Four months later when she came for the elective surgery, her pre transfusion investigations showed blood group as B Rh D positive, with a probable R1R1 phenotype. Her antibody screening was positive in all the three panel cells. Further testings showed a negative Direct Coomb's test and negative autocontrol, antibody identification showed pan-agglutination reaction on all 11 panel cells with enzyme enhancement. Patient's red cell phenotype was Jk(a-b-). Anti-Jk3 was suspected and further confirmed in the reference laboratory by phenotyping as well as negative urea lysis test. This case report highlights an extremely rare but clinically significant anti-JK3 antibody detected during pretransfusion testing. This phenotype is rare in the white population, more commonly seen in various polynesians. Increased awareness among the blood bank personnel regarding the variability of the blood group phenotype and the capricious nature of the Kidd antibodies may contribute to the better management of these patients.

Mucosal BCG Vaccination Induces Protective Lung-Resident Memory T Cell Populations against Tuberculosis

PubMed Central

Perdomo, Carolina; Zedler, Ulrike; Kühl, Anja A.; Lozza, Laura; Saikali, Philippe; Sander, Leif E.; Vogelzang, Alexis; Kupz, Andreas

2016-01-01

ABSTRACT Mycobacterium bovis Bacille Calmette-Guérin (BCG) is the only licensed vaccine against tuberculosis (TB), yet its moderate efficacy against pulmonary TB calls for improved vaccination strategies. Mucosal BCG vaccination generates superior protection against TB in animal models; however, the mechanisms of protection remain elusive. Tissue-resident memory T (TRM) cells have been implicated in protective immune responses against viral infections, but the role of TRM cells following mycobacterial infection is unknown. Using a mouse model of TB, we compared protection and lung cellular infiltrates of parenteral and mucosal BCG vaccination. Adoptive transfer and gene expression analyses of lung airway cells were performed to determine the protective capacities and phenotypes of different memory T cell subsets. In comparison to subcutaneous vaccination, intratracheal and intranasal BCG vaccination generated T effector memory and TRM cells in the lung, as defined by surface marker phenotype. Adoptive mucosal transfer of these airway-resident memory T cells into naive mice mediated protection against TB. Whereas airway-resident memory CD4+ T cells displayed a mixture of effector and regulatory phenotype, airway-resident memory CD8+ T cells displayed prototypical TRM features. Our data demonstrate a key role for mucosal vaccination-induced airway-resident T cells in the host defense against pulmonary TB. These results have direct implications for the design of refined vaccination strategies. PMID:27879332

Photofrin binds to procaspase-3 and mediates photodynamic treatment-triggered methionine oxidation and inactivation of procaspase-3

PubMed Central

Hsieh, Y-J; Chien, K-Y; Lin, S-Y; Sabu, S; Hsu, R-M; Chi, L-M; Lyu, P-C; Yu, J-S

2012-01-01

Diverse death phenotypes of cancer cells can be induced by Photofrin-mediated photodynamic therapy (PDT), which has a decisive role in eliciting a tumor-specific immunity for long-term tumor control. However, the mechanism(s) underlying this diversity remain elusive. Caspase-3 is a critical factor in determining cell death phenotypes in many physiological settings. Here, we report that Photofrin-PDT can modify and inactivate procaspase-3 in cancer cells. In cells exposed to an external apoptotic trigger, high-dose Photofrin-PDT pretreatment blocked the proteolytic activation of procaspase-3 by its upstream caspase. We generated and purified recombinant procaspase-3-D3A (a mutant without autolysis/autoactivation activity) to explore the underlying mechanism(s). Photofrin could bind directly to procaspase-3-D3A, and Photofrin-PDT-triggered inactivation and modification of procaspase-3-D3A was seen in vitro. Mass spectrometry-based quantitative analysis for post-translational modifications using both 16O/18O- and 14N/15N-labeling strategies revealed that Photofrin-PDT triggered a significant oxidation of procaspase-3-D3A (mainly on Met-27, -39 and -44) in a Photofrin dose-dependent manner, whereas the active site Cys-163 remained largely unmodified. Site-directed mutagenesis experiments further showed that Met-44 has an important role in procaspase-3 activation. Collectively, our results reveal that Met oxidation is a novel mechanism for the Photofrin-PDT-mediated inactivation of procaspase-3, potentially explaining at least some of the complicated cell death phenotypes triggered by PDT. PMID:22785533

Epithelial-mesenchymal transition and tumor suppression are controlled by a reciprocal feedback loop between ZEB1 and Grainyhead-like-2

PubMed Central

Cieply, Benjamin; Farris, Joshua; Denvir, James; Ford, Heide; Frisch, Steven M.

2013-01-01

Epithelial-mesenchymal transition (EMT) in carcinoma cells enhances malignant progression by promoting invasion and survival. EMT is induced by microenvironmental factors including TGF-β and Wnt agonists, and by the E-box-binding transcription factors Twist, Snail and ZEB. Grainyhead-like-2 (GRHL2), a member of the mammalian Grainyhead family of wound healing regulatory transcription factors, suppresses EMT and restores sensitivity to anoikis by repressing ZEB1 expression and inhibiting TGF-β signaling. In this study, we elucidate the functional relationship between GRHL2 and ZEB1 in EMT/MET and tumor biology. At least three homeodomain proteins, Six1, LBX1, and HoxA5, transactivated the ZEB1 promoter, in the case of Six1, through direct protein-promoter interaction. GRHL2 altered the Six1-DNA complex, inhibiting this transactivation. Correspondingly, GRHL2 expression prevented tumor initiation in xenograft assays, sensitized breast cancer cells to paclitaxel and suppressed the emergence of CD44highCD24low cells (defining the cancer stem cell phenotype in the cell type studied). GRHL2 was down-regulated in recurrent mouse tumors that had evolved to an oncogene-independent, EMT-like state, supporting a role for GRHL2 down-regulation in this phenotypic transition, modeling disease recurrence. The combination of TGF-β and Wnt activation repressed GRHL2 expression by direct interaction of ZEB1 with the GRHL2 promoter, inducing EMT. Together, our observations indicate that a reciprocal feedback loop between GRHL2 and ZEB1 controls epithelial vs. mesenchymal phenotypes and EMT-driven tumor progression. PMID:23943797

Modulation of the malignant phenotype with the urokinase-type plasminogen activator and the type I plasminogen activator inhibitor.

PubMed

Sordat, B; Reiter, L; Cajot, J F

1990-12-02

Gene transfer techniques were utilized to evaluate the role of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) in enhancing or preventing the expression of the invasive malignant phenotype, respectively. Mouse L-cell transfectants expressing human uPA or human PAI-1 as well as mouse B16 transfectants expressing mouse uPA or human PAI-1 were generated. These transfectants were tested using a variety of experimental methods including smooth muscle cell matrix solubilization in vitro, lung colony formation in vivo and co-cultures of antagonist-expressing cells in vitro. Results from these studies provide direct evidence for an enhancing role of uPA in malignant invasion and experimental metastasis and for a modulatory role of PAI-1 in tumor cell-mediated breakdown of extracellular matrices.

Complete Reconstitution of the Vancomycin-Intermediate Staphylococcus aureus Phenotype of Strain Mu50 in Vancomycin-Susceptible S. aureus

PubMed Central

Sekine, Miwa; Hishinuma, Tomomi; Aiba, Yoshifumi; Hiramatsu, Keiichi

2016-01-01

Complete reconstitution of the vancomycin-intermediate Staphylococcus aureus (VISA) phenotype of strain Mu50 was achieved by sequentially introducing mutations into six genes of vancomycin-susceptible S. aureus (VSSA) strain N315ΔIP. The six mutated genes were detected in VISA strain Mu50 but not in N315ΔIP. Introduction of the mutation Ser329Leu into vraS, encoding the sensor histidine kinase of the vraSR two-component regulatory (TCR) system, and another mutation, Glu146Lys, into msrR, belonging to the LytR-CpsA-Psr (LCP) family, increased the level of vancomycin resistance to that detected in heterogeneous vancomycin-intermediate S. aureus (hVISA) strain Mu3. Introduction of two more mutations, Asn197Ser into graR of the graSR TCR system and His481Tyr into rpoB, encoding the β subunit of RNA polymerase, converted the hVISA strain into a VISA strain with the same level of vancomycin resistance as Mu50. Surprisingly, however, the constructed quadruple mutant strain ΔIP4 did not have a thickened cell wall, a cardinal feature of the VISA phenotype. Subsequent study showed that cell wall thickening was an inducible phenotype in the mutant strain, whereas it was a constitutive one in Mu50. Finally, introduction of the Ala297Val mutation into fdh2, which encodes a putative formate dehydrogenase, or a 67-amino-acid sequence deletion into sle1 [sle1(Δ67aa)], encoding the hydrolase of N-acetylmuramyl-l-alanine amidase in the peptidoglycan, converted inducible cell wall thickening into constitutive cell wall thickening. sle1(Δ67aa) was found to cause a drastic decrease in autolysis activity. Thus, all six mutated genes required for acquisition of the VISA phenotype were directly or indirectly involved in the regulation of cell physiology. The VISA phenotype seemed to be achieved through multiple genetic events accompanying drastic changes in cell physiology. PMID:27067329

Generation and customization of biosynthetic excitable tissues for electrophysiological studies and cell-based therapies.

PubMed

Nguyen, Hung X; Kirkton, Robert D; Bursac, Nenad

2018-05-01

We describe a two-stage protocol to generate electrically excitable and actively conducting cell networks with stable and customizable electrophysiological phenotypes. Using this method, we have engineered monoclonally derived excitable tissues as a robust and reproducible platform to investigate how specific ion channels and mutations affect action potential (AP) shape and conduction. In the first stage of the protocol, we combine computational modeling, site-directed mutagenesis, and electrophysiological techniques to derive optimal sets of mammalian and/or prokaryotic ion channels that produce specific AP shape and conduction characteristics. In the second stage of the protocol, selected ion channels are stably expressed in unexcitable human cells by means of viral or nonviral delivery, followed by flow cytometry or antibiotic selection to purify the desired phenotype. This protocol can be used with traditional heterologous expression systems or primary excitable cells, and application of this method to primary fibroblasts may enable an alternative approach to cardiac cell therapy. Compared with existing methods, this protocol generates a well-defined, relatively homogeneous electrophysiological phenotype of excitable cells that facilitates experimental and computational studies of AP conduction and can decrease arrhythmogenic risk upon cell transplantation. Although basic cell culture and molecular biology techniques are sufficient to generate excitable tissues using the described protocol, experience with patch-clamp techniques is required to characterize and optimize derived cell populations.

FOXP3 Expression in GARP-Transduced Helper T Cells Is Not Associated with FOXP3 TSDR Demethylation

PubMed Central

Kehrmann, Jan; Zeschnigk, Michael; Buer, Jan; Probst-Kepper, Michael

2011-01-01

Aim: Glycoprotein A repetitions predominant (GARP or LRRC32) represents a human regulatory CD4+ CD25hi FOXP3+ T (Treg) cell-specific receptor that controls FOXP3. Ectopic expression of GARP in helper T (Th) cells has been shown to be sufficient for the induction of FOXP3 and generation of a stable regulatory phenotype. Since expression of FOXP3 in Treg cells is epigenetically controlled by a conserved motif, the so-called Treg-specific demethylated region (TSDR), we asked whether GARP-mediated upregulation of FOXP3 in Th cells is similarly accompanied by demethylation of the TSDR. Methods: DNA methylation of the FOXP3 TSDR was analyzed by direct sequencing of polymerase chain reaction (PCR) products from bisulfite-treated genomic DNA. Results: Although GARP-transduced Th cells exhibit constitutive FOXP3 expression and a regulatory phenotype, the FOXP3 TSDR is completely methylated as in naive Th cells. GARP-mediated FOXP3 upregulation in Th cells is not associated with Treg-specific demethylation of the FOXP3 TSDR. Conclusion: Although GARP-engineered Th cells exhibit stable FOXP3 expression and a phenotypic reprogramming towards Treg cells in vitro, these cells do not completely mimic the epigenotype of natural Treg cells. Thus, concepts based on the genetic modification of Th cells as cellular therapies to treat autoimmune diseases or to control transplantation tolerance should be critically tested before any clinical application. PMID:22670117

FOXP3 Expression in GARP-Transduced Helper T Cells Is Not Associated with FOXP3 TSDR Demethylation.

PubMed

Kehrmann, Jan; Zeschnigk, Michael; Buer, Jan; Probst-Kepper, Michael

2011-10-01

AIM: Glycoprotein A repetitions predominant (GARP or LRRC32) represents a human regulatory CD4+ CD25(hi) FOXP3+ T (T(reg)) cell-specific receptor that controls FOXP3. Ectopic expression of GARP in helper T (T(h)) cells has been shown to be sufficient for the induction of FOXP3 and generation of a stable regulatory phenotype. Since expression of FOXP3 in Treg cells is epigenetically controlled by a conserved motif, the so-called T(reg)-specific demethylated region (TSDR), we asked whether GARP-mediated upregulation of FOXP3 in Th cells is similarly accompanied by demethylation of the TSDR. METHODS: DNA methylation of the FOXP3 TSDR was analyzed by direct sequencing of polymerase chain reaction (PCR) products from bisulfite-treated genomic DNA. RESULTS: Although GARP-transduced T(h) cells exhibit constitutive FOXP3 expression and a regulatory phenotype, the FOXP3 TSDR is completely methylated as in naive T(h) cells. GARP-mediated FOXP3 upregulation in T(h) cells is not associated with T(reg)-specific demethylation of the FOXP3 TSDR. CONCLUSION: Although GARP-engineered T(h) cells exhibit stable FOXP3 expression and a phenotypic reprogramming towards T(reg) cells in vitro, these cells do not completely mimic the epigenotype of natural T(reg) cells. Thus, concepts based on the genetic modification of T(h) cells as cellular therapies to treat autoimmune diseases or to control transplantation tolerance should be critically tested before any clinical application.

CD8+ T Cells Primed in the Periphery Provide Time-Bound Immune-Surveillance to the Central Nervous System

PubMed Central

Young, Kevin G.; MacLean, Susanne; Dudani, Renu; Krishnan, Lakshmi; Sad, Subash

2016-01-01

After vaccination, memory CD8+ T cells migrate to different organs to mediate immune surveillance. In most nonlymphoid organs, following an infection, CD8+ T cells differentiate to become long-lived effector-memory cells, thereby providing long-term protection against a secondary infection. In this study, we demonstrated that Ag-specific CD8+ T cells that migrate to the mouse brain following a systemic Listeria infection do not display markers reminiscent of long-term memory cells. In contrast to spleen and other nonlymphoid organs, none of the CD8+ T cells in the brain reverted to a memory phenotype, and all of the cells were gradually eliminated. These nonmemory phenotype CD8+ T cells were found primarily within the choroid plexus, as well as in the cerebrospinal fluid-filled spaces. Entry of these CD8+ T cells into the brain was governed primarily by CD49d/VCAM-1, with the majority of entry occurring in the first week postinfection. When CD8+ T cells were injected directly into the brain parenchyma, cells that remained in the brain retained a highly activated (CD69hi) phenotype and were gradually lost, whereas those that migrated out to the spleen were CD69low and persisted long-term. These results revealed a mechanism of time-bound immune surveillance to the brain by CD8+ T cells that do not reside in the parenchyma. PMID:21715683

Rapid fusion between mesenchymal stem cells and cardiomyocytes yields electrically active, non-contractile hybrid cells.

PubMed

Shadrin, Ilya Y; Yoon, Woohyun; Li, Liqing; Shepherd, Neal; Bursac, Nenad

2015-07-10

Cardiac cell therapies involving bone marrow-derived human mesenchymal stem cells (hMSCs) have shown promising results, although their mechanisms of action are still poorly understood. Here, we investigated direct interactions between hMSCs and cardiomyocytes in vitro. Using a genetic Ca(2+) indicator gCaMP3 to efficiently label hMSCs in co-cultures with neonatal rat ventricular myocytes (NRVMs), we determined that 25-40% of hMSCs (from 4 independent donors) acquired periodic Ca(2+) transients and cardiac markers through spontaneous fusion with NRVMs. Sharp electrode and voltage-clamp recordings in fused cells showed action potential properties and Ca(2+) current amplitudes in between those of non-fused hMSCs and NRVMs. Time-lapse video-microscopy revealed the first direct evidence of active fusion between hMSCs and NRVMs within several hours of co-culture. Application of blebbistatin, nifedipine or verapamil caused complete and reversible inhibition of fusion, suggesting potential roles for actomyosin bridging and Ca(2+) channels in the fusion process. Immunostaining for Cx43, Ki67, and sarcomeric α-actinin showed that fused cells remain strongly coupled to surrounding NRVMs, but downregulate sarcomeric structures over time, acquiring a non-proliferative and non-contractile phenotype. Overall, these results describe the phenotype and mechanisms of hybrid cell formation via fusion of hMSCs and cardiomyocytes with potential implications for cardiac cell therapy.

Electrical and Mechanical Strategies to Enable Cardiac Repair and Regeneration

PubMed Central

Cao, Hung; Kang, Bong Jin; Lee, Chia-An; Shung, K. Kirk; Hsiai, Tzung K.

2015-01-01

Inadequate replacement of lost ventricular myocardium from myocardial infarction leads to heart failure. Investigating the regenerative capacity of mammalian hearts represents an emerging direction for tissue engineering and cell-based therapy. Recent advances in stem cells hold promise to restore cardiac functions. However, embryonic or induced pluripotent stem cell-derived cardiomyocytes lack functional phenotypes of the native myocardium, and transplanted tissues are not fully integrated for synchronized electrical and mechanical coupling with the host. In this context, this review highlights the mechanical and electrical strategies to promote cardiomyocyte maturation and integration, and to assess the functional phenotypes of regenerating myocardium. Simultaneous micro-electrocardiogram and high-frequency ultrasound techniques will also be introduced to assess electrical and mechanical coupling for small animal models of heart regeneration. PMID:25974948

Regulation of cell wall morphogenesis in Bacillus subtilis by recruitment of PBP1 to the MreB helix.

PubMed

Kawai, Yoshikazu; Daniel, Richard A; Errington, Jeffery

2009-03-01

The bacterial actin homologue MreB plays a key role in cell morphogenesis. In Bacillus subtilis MreB is essential under normal growth conditions and mreB mutants are defective in the control of cell diameter. However, the precise role of MreB is still unclear. Analysis of the lethal phenotypic consequences of mreB disruption revealed an unusual bulging phenotype that precedes cell death. A similar phenotype was seen in wild-type cells at very low Mg(2+) concentrations. We found that inactivation of the major bi-functional penicillin-binding protein (PBP) PBP1 of B. subtilis restored the viability of an mreB null mutant as well as preventing bulging in both mutant and wild-type backgrounds. Bulging was associated with delocalization of PBP1. We show that the normal pattern of localization of PBP1 is dependent on MreB and that the proteins can physically interact using in vivo pull-down and bacterial two-hybrid approaches. Interactions between MreB and several other PBPs were also detected. Our results suggest that MreB filaments associate directly with the peptidoglycan biosynthetic machinery in B. subtilis as part of the mechanism that brings about controlled cell elongation.

Direct Correlation between Motile Behavior and Protein Abundance in Single Cells

PubMed Central

Gillet, Sébastien; Frankel, Nicholas W.; Weibel, Douglas B.

2016-01-01

Understanding how stochastic molecular fluctuations affect cell behavior requires the quantification of both behavior and protein numbers in the same cells. Here, we combine automated microscopy with in situ hydrogel polymerization to measure single-cell protein expression after tracking swimming behavior. We characterized the distribution of non-genetic phenotypic diversity in Escherichia coli motility, which affects single-cell exploration. By expressing fluorescently tagged chemotaxis proteins (CheR and CheB) at different levels, we quantitatively mapped motile phenotype (tumble bias) to protein numbers using thousands of single-cell measurements. Our results disagreed with established models until we incorporated the role of CheB in receptor deamidation and the slow fluctuations in receptor methylation. Beyond refining models, our central finding is that changes in numbers of CheR and CheB affect the population mean tumble bias and its variance independently. Therefore, it is possible to adjust the degree of phenotypic diversity of a population by adjusting the global level of expression of CheR and CheB while keeping their ratio constant, which, as shown in previous studies, confers functional robustness to the system. Since genetic control of protein expression is heritable, our results suggest that non-genetic diversity in motile behavior is selectable, supporting earlier hypotheses that such diversity confers a selective advantage. PMID:27599206

Direct Reprogramming of Murine Fibroblasts to Hematopoietic Progenitor Cells

PubMed Central

Batta, Kiran; Florkowska, Magdalena; Kouskoff, Valerie; Lacaud, Georges

2014-01-01

Summary Recent reports have shown that somatic cells, under appropriate culture conditions, could be directly reprogrammed to cardiac, hepatic, or neuronal phenotype by lineage-specific transcription factors. In this study, we demonstrate that both embryonic and adult somatic fibroblasts can be efficiently reprogrammed to clonal multilineage hematopoietic progenitors by the ectopic expression of the transcription factors ERG, GATA2, LMO2, RUNX1c, and SCL. These reprogrammed cells were stably expanded on stromal cells and possessed short-term reconstitution ability in vivo. Loss of p53 function facilitated reprogramming to blood, and p53−/− reprogrammed cells efficiently generated erythroid, megakaryocytic, myeloid, and lymphoid lineages. Genome-wide analyses revealed that generation of hematopoietic progenitors was preceded by the appearance of hemogenic endothelial cells expressing endothelial and hematopoietic genes. Altogether, our findings suggest that direct reprogramming could represent a valid alternative approach to the differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) for disease modeling and autologous blood cell therapies. PMID:25466247

Critical high-dimensional state transitions in cell populations or why cancers follow the principle ``What does not kill me makes me stronger''

NASA Astrophysics Data System (ADS)

Huang, Sui

Transitions between high-dimensional attractor states in the quasi-potential landscape of the gene regulatory network, induced by environmental perturbations and/or facilitated by mutational rewiring of the network, underlie cell phenotype switching in development as well as in cancer progression, including acquisition of drug-resistant phenotypes. Considering heterogeneous cell populations as statistical ensembles of cells, and single-cell resolution gene expression profiling of cell populations undergoing a cell phenotype shift allow us now to map the topography of the landscape and its distortion. From snapshots of single-cell expression patterns of a cell population measured during major transitions we compute a quantity that identifies symmetry-breaking destabilization of attractors (bifurcation) and concomitant dimension-reduction of the state space manifold (landscape distortion) which precede critical transitions to new attractor states. The model predicts, and we show experimentally, the almost inevitable generation of aberrant cells associated with such critical transitions in multi-attractor landscapes: therapeutic perturbations which seek to push cancer cells to the apoptotic state, almost always produce ``rebellious'' cells which move in the ``opposite direction'': instead of dying they become more stem-cell-like and malignant. We show experimentally that the inadvertent generation of more malignant cancer cells by therapy indeed results from transition of surviving (but stressed) cells into unforeseen attractor states and not simply from selection of inherently more resistant cells. Thus, cancer cells follow not so much Darwin, as generally thought (survival of the fittest), but rather Nietzsche (What does not kill me makes me stronger). Supported by NIH (NCI, NIGMS), Alberta Innovates.

Rapamycin resistant murine th9 cells have a stable in vivo phenotype and inhibit graft-versus-host reactivity.

PubMed

Mangus, Courtney W; Massey, Paul R; Fowler, Daniel H; Amarnath, Shoba

2013-01-01

The cytokine micro-environment can direct murine CD4(+) T cells towards various differentiation lineages such as Th1, Th2 and Tregs even in the presence of rapamycin, which results in T cells that mediate increased in vivo effects. Recently, a new lineage of T cells known as Th9 cells that secrete increased IL-9 have been described. However, it is not known whether Th9 differentiation occurs in the presence of rapamycin or whether adoptively transferred donor Th9 cells would augment or restrict alloreactivity after experimental bone marrow transplantation. We found that CD4(+) T cells that were co-stimulated and polarized with TGF-β and IL-4 in the presence or absence of rapamycin each yielded effector cells of Th9 phenotype that secreted increased IL-9 and expressed a transcription factor profile characteristic of both Th9 and Th2 cells (high GATA-3/low T-bet). Augmentation of T cell replete allografts with manufactured rapamycin resistant Th9 cells markedly reduced both CD4(+) and CD8(+) T cell engraftment and strongly inhibited allo-specific T cell secretion of IFN-γ. The potency of Th9 cell inhibition of alloreactivity was similar to that of rapamycin resistant Th2 cells. Importantly, rapamycin resistant Th9 cells persisted and maintained their cytokine phenotype, thereby indicating limited differentiation plasticity of the Th9 subset. As such, Th9 differentiation proceeds in the presence of rapamycin to generate a cell therapy product that maintains high IL-9 expression in vivo while inhibiting IFN-γ driven alloreactivity.

Human brain microvascular endothelial cells resist elongation due to shear stress.

PubMed

Reinitz, Adam; DeStefano, Jackson; Ye, Mao; Wong, Andrew D; Searson, Peter C

2015-05-01

Endothelial cells in straight sections of vessels are known to elongate and align in the direction of flow. This phenotype has been replicated in confluent monolayers of bovine aortic endothelial cells and human umbilical vein endothelial cells (HUVECs) in cell culture under physiological shear stress. Here we report on the morphological response of human brain microvascular endothelial cells (HBMECs) in confluent monolayers in response to shear stress. Using a microfluidic platform we image confluent monolayers of HBMECs and HUVECs under shear stresses up to 16 dyne cm(-2). From live-cell imaging we quantitatively analyze the cell morphology and cell speed as a function of time. We show that HBMECs do not undergo a classical transition from cobblestone to spindle-like morphology in response to shear stress. We further show that under shear stress, actin fibers are randomly oriented in the cells indicating that there is no cytoskeletal remodeling. These results suggest that HBMECs are programmed to resist elongation and alignment under shear stress, a phenotype that may be associated with the unique properties of the blood-brain barrier. Copyright © 2015 Elsevier Inc. All rights reserved.

Finite element modeling predictions of region-specific cell-matrix mechanics in the meniscus.

PubMed

Upton, Maureen L; Guilak, Farshid; Laursen, Tod A; Setton, Lori A

2006-06-01

The knee meniscus exhibits significant spatial variations in biochemical composition and cell morphology that reflect distinct phenotypes of cells located in the radial inner and outer regions. Associated with these cell phenotypes is a spatially heterogeneous microstructure and mechanical environment with the innermost regions experiencing higher fluid pressures and lower tensile strains than the outer regions. It is presently unknown, however, how meniscus tissue mechanics correlate with the local micromechanical environment of cells. In this study, theoretical models were developed to study mechanics of inner and outer meniscus cells with varying geometries. The results for an applied biaxial strain predict significant regional differences in the cellular mechanical environment with evidence of tensile strains along the collagen fiber direction of approximately 0.07 for the rounded inner cells, as compared to levels of 0.02-0.04 for the elongated outer meniscus cells. The results demonstrate an important mechanical role of extracellular matrix anisotropy and cell morphology in regulating the region-specific micromechanics of meniscus cells, that may further play a role in modulating cellular responses to mechanical stimuli.

An in-vitro scaffold-free epithelial-fibroblast coculture model for the larynx

PubMed Central

Walimbe, Tanaya; Panitch, Alyssa; Sivasankar, M. Preeti

2017-01-01

Objective Physiologically relevant, well-characterized in vitro vocal fold coculture models are needed to test the effects of various challenges and therapeutics on vocal fold physiology. We characterize a healthy state coculture model, created by using bronchial/tracheal epithelial cells and immortalized vocal fold fibroblasts. We also demonstrate that this model can be induced into a fibroplastic state to overexpress stress fibers using TGFβ1. Method Cell metabolic activity of immortalized human vocal fold fibroblasts incubated in different media combinations were confirmed with MTT assay. Fibroblasts were grown to confluence and primary bronchial/tracheal epithelial cells suspended in coculture media were seeded directly over the base layer of the fibroblasts. Cells were treated with TGFβ1 to induce myofibroblast formation. Cell shape and position was confirmed by live cell tracking, fibrosis was confirmed by probing for α smooth muscle actin (α-SMA) and phenotype was confirmed by immunostaining for vimentin and E-cadherin. Results Fibroblasts retain metabolic activity in coculture epithelial media. Live cell imaging revealed a layer of epithelial cells atop fibroblasts. α-SMA expression was enhanced in TGFβ1 treated cells, confirming that both cell types maintained a healthy phenotype in coculture, and can be induced into overexpressing stress fibers. Vimentin and E-cadherin immunostaining show that cells retain phenotype in coculture. Conclusion These data lay effective groundwork for a functional coculture model that retains the reproducibility necessary to serve as a viable diagnostic and therapeutic screening platform. Level of Evidence NA PMID:27859361

Mesenchymal Stem Cells Sense Three Dimensional Type I Collagen through Discoidin Domain Receptor 1.

PubMed

Lund, A W; Stegemann, J P; Plopper, G E

2009-01-01

The extracellular matrix provides structural and organizational cues for tissue development and defines and maintains cellular phenotype during cell fate determination. Multipotent mesenchymal stem cells use this matrix to tightly regulate the balance between their differentiation potential and self-renewal in the native niche. When understood, the mechanisms that govern cell-matrix crosstalk during differentiation will allow for efficient engineering of natural and synthetic matrices to specifically direct and maintain stem cell phenotype. This work identifies the discoidin domain receptor 1 (DDR1), a collagen activated receptor tyrosine kinase, as a potential link through which stem cells sense and respond to the 3D organization of their extracellular matrix microenvironment. DDR1 is dependent upon both the structure and proteolytic state of its collagen ligand and is specifically expressed and localized in three dimensional type I collagen culture. Inhibition of DDR1 expression results in decreased osteogenic potential, increased cell spreading, stress fiber formation and ERK1/2 phosphorylation. Additionally, loss of DDR1 activity alters the cell-mediated organization of the naïve type I collagen matrix. Taken together, these results demonstrate a role for DDR1 in the stem cell response to and interaction with three dimensional type I collagen. Dynamic changes in cell shape in 3D culture and the tuning of the local ECM microstructure, directs crosstalk between DDR1 and two dimensional mechanisms of osteogenesis that can alter their traditional roles.

Induction of CaSR expression circumvents the molecular features of malignant CaSR null colon cancer cells.

PubMed

Singh, Navneet; Chakrabarty, Subhas

2013-11-15

We recently reported on the isolation and characterization of calcium sensing receptor (CaSR) null human colon cancer cells (Singh et al., Int J Cancer 2013; 132: 1996-2005). CaSR null cells possess a myriad of molecular features that are linked to a highly malignant and drug resistant phenotype of colon cancer. The CaSR null phenotype can be maintained in defined human embryonic stem cell culture medium. We now show that the CaSR null cells can be induced to differentiate in conventional culture medium, regained the expression of CaSR with a concurrent reversal of the cellular and molecular features associated with the null phenotype. These features include cellular morphology, expression of colon cancer stem cell markers, expression of survivin and thymidylate synthase and sensitivity to fluorouracil. Other features include the expression of epithelial mesenchymal transition linked molecules and transcription factors, oncogenic miRNAs and tumor suppressive molecule and miRNA. With the exception of cancer stem cell markers, the reversal of molecular features, upon the induction of CaSR expression, is directly linked to the expression and function of CaSR because blocking CaSR induction by shRNA circumvented such reversal. We further report that methylation and demethylation of the CaSR gene promoter underlie CaSR expression. Due to the malignant nature of the CaSR null cells, inclusion of the CaSR null phenotype in disease management may improve on the mortality of this disease. Because CaSR is a robust promoter of differentiation and mediates its action through diverse mechanisms and pathways, inactivation of CaSR may serve as a new paradigm in colon carcinogenesis. Copyright © 2013 UICC.

Functional differentiation of human pluripotent stem cells on a chip.

PubMed

Giobbe, Giovanni G; Michielin, Federica; Luni, Camilla; Giulitti, Stefano; Martewicz, Sebastian; Dupont, Sirio; Floreani, Annarosa; Elvassore, Nicola

2015-07-01

Microengineering human "organs-on-chips" remains an open challenge. Here, we describe a robust microfluidics-based approach for the differentiation of human pluripotent stem cells directly on a chip. Extrinsic signal modulation, achieved through optimal frequency of medium delivery, can be used as a parameter for improved germ layer specification and cell differentiation. Human cardiomyocytes and hepatocytes derived on chips showed functional phenotypes and responses to temporally defined drug treatments.

Overexpression of SDF-1 activates the NF-κB pathway to induce epithelial to mesenchymal transition and cancer stem cell-like phenotypes of breast cancer cells.

PubMed

Kong, Lingxin; Guo, Sufen; Liu, Chunfeng; Zhao, Yiling; Feng, Chong; Liu, Yunshuang; Wang, Tao; Li, Caijuan

2016-03-01

The formation of EMT and EMT-induced CSC-like phenotype is crucial for the metastasis of tumor cells. The stromal cell-derived factor-1 (SDF-1) is upregulated in various human carcinomas, which is closely associated with proliferation, migration, invasion and prognosis of malignancies. However, limited attention has been directed towards the effect of SDF-1 on epithelial to mesenchymal transition (EMT) or cancer stem cell (CSC)-like phenotype formation in breast cancer cells and the related mechanism. In the present study, we screened MCF-7 cells with low SDF-1 expression level for the purpose of evaluating whether SDF-1 is involved in EMT and CSC-like phenotype formation in MCF-7 cells. The pEGFP-N1-SDF-1 plasmid was transfected into MCF-7 cells, and the stably overexpressed SDF-1 in MCF-7 cells was confirmed by real-time PCR and western blot analysis. Colony formation assay, MTT, wound healing assay and Transwell invasion assay demonstrated that overexpression of SDF-1 significantly boosted the proliferation, migration and invasion of MCF-7 cells compared with parental (P<0.05). Flow cytometry analysis revealed a notable increase of CD44+/CD24- subpopulation in SDF-1 overexpressing MCF-7 cells (P<0.001), accompanied by the apparently elevated ALDH activity and the upregulation of the stem cell markers OCT-4, Nanog, and SOX2 compared with parental (P<0.01). Besides, western blot analysis and immunofluorescence assay observed the significant decreased expression of E-cadherin and enhanced expression of slug, fibronectin and vimentin in SDF-1 overexpressed MCF-7 cells in comparison with parental (P<0.01). Further study found that overexpression of SDF-1 induced the activation of NF-κB pathway in MCF-7 cells. Conversely, suppressing or silencing p65 expression by antagonist or RNA interference could remarkably increase the expression of E-cadherin in SDF-1 overexpressed MCF-7 cells (P<0.001). Overall, the above results indicated that overexpression of SDF-1 enhanced EMT by activating the NF-κB pathway of MCF-7 cells and further induced the formation of CSC-like phenotypes, ultimately promoting the proliferation and metastasis of MCF-7 cells. Therefore, SDF-1 may further be assessed as a potential target for gene therapy of breast cancer.

Hybridization and Polyploidization of Saccharomyces cerevisiae Strains by Transformation-Associated Cell Fusion

PubMed Central

Takagi, Atsuko; Harashima, Satoshi; Oshima, Yasuji

1985-01-01

Hybrid or polyploid clones of Saccharomyces cerevisiae produced by protoplast fusion were easily isolated by selecting transformants with the plasmid phenotype because the transformation was directly associated with cell fusion. When haploid cells were used as the original strain, the transformants were mostly diploids with a significant fraction of polyploids (triploids or tetraploids). Repeated transformation after curing the plasmid gave rise to clones with higher ploidy, but the frequency of cell fusion was severely reduced as ploidy increased. Images PMID:16346702

Gut-derived factors promote neurogenesis of CNS-neural stem cells and nudge their differentiation to an enteric-like neuronal phenotype.

PubMed

Kulkarni, Subhash; Zou, Bende; Hanson, Jesse; Micci, Maria-Adelaide; Tiwari, Gunjan; Becker, Laren; Kaiser, Martin; Xie, Xinmin Simon; Pasricha, Pankaj Jay

2011-10-01

Recent studies have explored the potential of central nervous system-derived neural stem cells (CNS-NSC) to repopulate the enteric nervous system. However, the exact phenotypic fate of gut-transplanted CNS-NSC has not been characterized. The aim of this study was to investigate the effect of the gut microenvironment on phenotypic fate of CNS-NSC in vitro. With the use of Transwell culture, differentiation of mouse embryonic CNS-NSC was studied when cocultured without direct contact with mouse intestinal longitudinal muscle-myenteric plexus preparations (LM-MP) compared with control noncocultured cells, in a differentiating medium. Differentiated cells were analyzed by immunocytochemistry and quantitative RT-PCR to assess the expression of specific markers and by whole cell patch-clamp studies for functional characterization of their phenotype. We found that LM-MP cocultured cells had a significant increase in the numbers of cells that were immune reactive against the panneuronal marker β-tubulin, neurotransmitters neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and neuropeptide vasoactive intestinal peptide (VIP) and showed an increase in expression of these genes, compared with control cells. Whole cell patch-clamp analysis showed that coculture with LM-MP decreases cell excitability and reduces voltage-gated Na(+) currents but significantly enhances A-current and late afterhyperpolarization (AHP) and increases the expression of the four AHP-generating Ca(2+)-dependent K(+) channel genes (KCNN), compared with control cells. In a separate experiment, differentiation of LM-MP cocultured CNS-NSC produced a significant increase in the numbers of cells that were immune reactive against the neurotransmitters nNOS, ChAT, and the neuropeptide VIP compared with CNS-NSC differentiated similarly in the presence of neonatal brain tissue. Our results show that the gut microenvironment induces CNS-NSC to produce neurons that share some of the characteristics of classical enteric neurons, further supporting the therapeutic use of these cells for gastrointestinal disorders.

Immune Checkpoint Blockade for Breast Cancer.

PubMed

Swoboda, April; Nanda, Rita

An effective antitumor immune response requires interaction between cells of the adaptive and innate immune system. Three key elements are required: generation of activated tumor-directed T cells, infiltration of activated T cells into the tumor microenvironment, and killing of tumor cells by activated T cells. Tumor immune evasion can occur as a result of the disruption of each of these three key T cell activities, resulting in three distinct cancer-immune phenotypes. The immune inflamed phenotype, characterized by the presence of a robust tumor immune infiltrate, suggests impaired activated T cell killing of tumor cells related to the presence of inhibitory factors. Programmed death receptor-1 (PD-1) is an inhibitory transmembrane protein expressed on T cells, B cells, and NK cells. The interaction between PD-1 and its ligands (PD-L1/L2) functions as an immune checkpoint against unrestrained cytotoxic T effector cell activity-it promotes peripheral T effector cell exhaustion and conversion of T effector cells to immunosuppressive T regulatory (Treg) cells. Immune checkpoint inhibitors, which block the PD-1/PD-L1 axis and reactivate cytotoxic T effector cell function, are actively being investigated for the treatment of breast cancer.

Functional and phenotypical analysis of IL-6-secreting CD4+ T cells in human adipose tissue.

PubMed

de Jong, Anja J; Pollastro, Sabrina; Kwekkeboom, Joanneke C; Andersen, Stefan N; Dorjée, Annemarie L; Bakker, Aleida M; Alzaid, Fawaz; Soprani, Antoine; Nelissen, Rob G H H; Mullers, Jan B; Venteclef, Nicolas; de Vries, Niek; Kloppenburg, Margreet; Toes, René E M; Ioan-Facsinay, Andreea

2018-03-01

Emerging evidence indicates that a dynamic interplay between the immune system and adipocytes contributes to the disturbed homeostasis in adipose tissue of obese subjects. Recently, we observed IL-6-secretion by CD4 + T cells from the stromal vascular fraction (SVF) of the infrapatellar fat pad (IFP) of knee osteoarthritis patients directly ex vivo. Here we show that human IL-6 + CD4 + T cells from SVF display a more activated phenotype than the IL-6 - T cells, as evidenced by the expression of the activation marker CD69. Analysis of cytokines secretion, as well as expression of chemokine receptors and transcription factors associated with different Th subsets (Treg, Th1, Th2, Th17 and Tfh) revealed that IL-6-secreting CD4 + T cells cannot be assigned to a conventional Th subset. TCRβ gene analysis revealed that IL-6 + and IL-6 - CD4 + T cells appear clonally unrelated to each other, suggesting a different specificity of these cells. In line with these observations, adipocytes are capable of enhancing IL-6 production by CD4 + T cells. Thus, IL-6 + CD4 + T cells are TCRαβ T cells expressing an activated phenotype potentially resulting from an interplay with adipocytes that could be involved in the inflammatory processes in the OA joint. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Iterative design of peptide-based hydrogels and the effect of network electrostatics on primary chondrocyte behavior

PubMed Central

Sinthuvanich, Chomdao; Haines-Butterick, Lisa A.; Nagy, Katelyn J.; Schneider, Joel P.

2012-01-01

Iterative peptide design was used to generate two peptide-based hydrogels to study the effect of network electrostatics on primary chondrocyte behavior. MAX8 and HLT2 peptides have formal charge states of +7 and +5 per monomer, respectively. These peptides undergo triggered folding and self-assembly to afford hydrogel networks having similar rheological behavior and local network morphologies, yet different electrostatic character. Each gel can be used to directly encapsulate and syringe-deliver cells. The influence of network electrostatics on cell viability after encapsulation and delivery, extracellular matrix deposition, gene expression, and the bulk mechanical properties of the gel-cell constructs as a function of culture time was assessed. The less electropositive HLT2 gel provides a microenvironment more conducive to chondrocyte encapsulation, delivery, and phenotype maintenance. Cell viability was higher for this gel and although a moderate number of cells dedifferentiated to a fibroblast-like phenotype, many retained their chondrocytic behavior. As a result, gel-cell constructs prepared with HLT2, cultured under static in vitro conditions, contained more GAG and type II collagen resulting in mechanically superior constructs. Chondrocytes delivered in the more electropositive MAX8 gel experienced a greater degree of cell death during encapsulation and delivery and the remaining viable cells were less prone to maintain their phenotype. As a result, MAX8 gel-cell constructs had fewer cells, of which a limited number were capable of laying down cartilage-specific ECM. PMID:22841922

Iterative design of peptide-based hydrogels and the effect of network electrostatics on primary chondrocyte behavior.

PubMed

Sinthuvanich, Chomdao; Haines-Butterick, Lisa A; Nagy, Katelyn J; Schneider, Joel P

2012-10-01

Iterative peptide design was used to generate two peptide-based hydrogels to study the effect of network electrostatics on primary chondrocyte behavior. MAX8 and HLT2 peptides have formal charge states of +7 and +5 per monomer, respectively. These peptides undergo triggered folding and self-assembly to afford hydrogel networks having similar rheological behavior and local network morphologies, yet different electrostatic character. Each gel can be used to directly encapsulate and syringe-deliver cells. The influence of network electrostatics on cell viability after encapsulation and delivery, extracellular matrix deposition, gene expression, and the bulk mechanical properties of the gel-cell constructs as a function of culture time was assessed. The less electropositive HLT2 gel provides a microenvironment more conducive to chondrocyte encapsulation, delivery, and phenotype maintenance. Cell viability was higher for this gel and although a moderate number of cells dedifferentiated to a fibroblast-like phenotype, many retained their chondrocytic behavior. As a result, gel-cell constructs prepared with HLT2, cultured under static in vitro conditions, contained more GAG and type II collagen resulting in mechanically superior constructs. Chondrocytes delivered in the more electropositive MAX8 gel experienced a greater degree of cell death during encapsulation and delivery and the remaining viable cells were less prone to maintain their phenotype. As a result, MAX8 gel-cell constructs had fewer cells, of which a limited number were capable of laying down cartilage-specific ECM. Published by Elsevier Ltd.

Brain nuclei in actively courting red-sided garter snakes: a paradigm of neural trimorphism.

PubMed

Krohmer, Randolph W; DeMarchi, Geno A; Baleckaitis, Daniel D; Lutterschmidt, Deborah I; Mason, Robert T

2011-03-28

During the breeding season, two distinct male phenotypes are exhibited by red-sided garter snakes (Thamnophis sirtalis parietalis), with courtship behavior being directed not only toward females, but also toward a sub-population of males called she-males. She-males are morphologically identical to other males except for a circulating androgen level three times that of normal males and their ability to produce a female-like pheromone. As in other vertebrates, limbic nuclei in the red-sided garter snake brain are involved in the control of sexual behaviors. For example, an intact anterior hypothalamus pre-optic area (AHPOA) is essential for the initiation and maintenance of reproduction. To determine if brain morphology varies among the three behavioral phenotypes (i.e., males, she-males, and females) during the breeding season, we examined the volume, cell size and cell density of the AHPOA as well as a control region, the external nucleus of the optic tract (ENOT). We used Luxol Fast Blue and Ziehl's Fuchsin to visualize neurons and glial cells, respectively. No significant differences were observed among the three behavioral phenotypes in the volume, cell size or density in the control region. In contrast, the volume, cell size and density of the AHPOA of she-males were significantly greater than those of both male and female snakes. While the volume of the AHPOA was significantly greater in females compared to males, no differences were observed in cell size or density. These differences in brain morphology suggest a possible underlying mechanism for phenotypic-specific behavioral patterns. Copyright © 2010 Elsevier Inc. All rights reserved.

Direct Reprogramming of Human Fibroblasts toward a Cardiomyocyte-like State

PubMed Central

Fu, Ji-Dong; Stone, Nicole R.; Liu, Lei; Spencer, C. Ian; Qian, Li; Hayashi, Yohei; Delgado-Olguin, Paul; Ding, Sheng; Bruneau, Benoit G.; Srivastava, Deepak

2013-01-01

Summary Direct reprogramming of adult somatic cells into alternative cell types has been shown for several lineages. We previously showed that GATA4, MEF2C, and TBX5 (GMT) directly reprogrammed nonmyocyte mouse heart cells into induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. However, GMT alone appears insufficient in human fibroblasts, at least in vitro. Here, we show that GMT plus ESRRG and MESP1 induced global cardiac gene-expression and phenotypic shifts in human fibroblasts derived from embryonic stem cells, fetal heart, and neonatal skin. Adding Myocardin and ZFPM2 enhanced reprogramming, including sarcomere formation, calcium transients, and action potentials, although the efficiency remained low. Human iCM reprogramming was epigenetically stable. Furthermore, we found that transforming growth factor β signaling was important for, and improved the efficiency of, human iCM reprogramming. These findings demonstrate that human fibroblasts can be directly reprogrammed toward the cardiac lineage, and lay the foundation for future refinements in vitro and in vivo. PMID:24319660

Transitions towards either slow-oxidative or fast-glycolytic phenotype can be induced in the murine WTt myogenic cell line.

PubMed

Peltzer, J; Carpentier, G; Martelly, I; Courty, J; Keller, A

2010-09-01

Contraction and energy metabolism are functions of skeletal muscles co-regulated by still largely unknown signals. To help elucidating these interconnecting pathways, we are developing new cellular models that will allow to control the switch from a neonatal to an adult slow-oxidative or fast-glycolytic phenotype of myofibers, during in vitro differentiation. Thus, our purpose was to direct the differentiation of the newly characterized WTt clone, from a mixed towards either fast or slow phenotype, by modifying amounts of two transcription factors respectively involved in control of glycolytic and oxidative energy metabolism, namely HIF-1alpha and PPARdelta. Our data support the idea that HIF-1alpha protein stabilization would favor expression of fast phenotypic markers, accompanied or not by a decreased expression of slow markers, depending on treatment conditions. Conversely, PPARdelta over-expression appears to enhance the slow-oxidative phenotype of WTt myotubes. Furthermore, we have observed that expression of PGC-1alpha, a coregulator of PPAR, is also modified in this cell line upon conditions that stabilize HIF-1alpha protein. This observation points to the existence of a regulatory link between pathways controlled by the two transcription factors HIF-1alpha and PPARdelta. Therefore, these cells should be useful to analyze the balance between oxidative and glycolytic energy production as a function of phenotypic transitions occurring during myogenic maturation. The newly characterized murine WTt clone will be a good tool to investigate molecular mechanisms implicating HIF-1alpha and PPARdelta in the coordinated metabolic and contractile regulations involved in myogenesis. (c) 2010 Wiley-Liss, Inc.

Donor-Dependent and Other Nondefined Factors Have Greater Influence on the Hepatic Phenotype Than the Starting Cell Type in Induced Pluripotent Stem Cell Derived Hepatocyte-Like Cells.

PubMed

Heslop, James A; Kia, Richard; Pridgeon, Christopher S; Sison-Young, Rowena L; Liloglou, Triantafillos; Elmasry, Mohamed; Fenwick, Stephen W; Mills, John S; Kitteringham, Neil R; Goldring, Chris E; Park, Bong K

2017-05-01

Drug-induced liver injury is the greatest cause of post-marketing drug withdrawal; therefore, substantial resources are directed toward triaging potentially dangerous new compounds at all stages of drug development. One of the major factors preventing effective screening of new compounds is the lack of a predictive in vitro model of hepatotoxicity. Primary human hepatocytes offer a metabolically relevant model for which the molecular initiating events of hepatotoxicity can be examined; however, these cells vary greatly between donors and dedifferentiate rapidly in culture. Induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) offer a reproducible, physiologically relevant and genotypically normal model cell; however, current differentiation protocols produce HLCs with a relatively immature phenotype. During the reprogramming of somatic cells, the epigenome undergoes dramatic changes; however, this "resetting" is a gradual process, resulting in an altered differentiation propensity, skewed toward the lineage of origin, particularly in early passage cultures. We, therefore, performed a comparison of human hepatocyte- and dermal fibroblast-derived iPSCs, assessing the impact of epigenetic memory at all stages of HLC differentiation. These results provide the first isogenic assessment of the starting cell type in human iPSC-derived HLCs. Despite a trend toward improvement in hepatic phenotype in albumin secretion and gene expression, few significant differences in hepatic differentiation capacity were found between hepatocyte and fibroblast-derived iPSCs. We conclude that the donor and inter-clonal differences have a greater influence on the hepatocyte phenotypic maturity than the starting cell type. Therefore, it is not necessary to use human hepatocytes for generating iPSC-derived HLCs. Stem Cells Translational Medicine 2017;6:1321-1331. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Uncovering cancer cell behavioral phenotype in 3-D in vitro metastatic landscapes

NASA Astrophysics Data System (ADS)

Liu, Liyu; Sun, Bo; Duclos, Guillaume; Kam, Yoonseok; Gatenby, Robert; Stone, Howard; Austin, Robert

2012-02-01

One well-known fact is that cancer cell genetics determines cell metastatic potentials. However, from a physics point of view, genetics as cell properties cannot directly act on metastasis. An agent is needed to unscramble the genetics first before generating dynamics for metastasis. Exactly this agent is cell behavioral phenotype, which is rarely studied due to the difficulties of real-time cell tracking in in vivo tissue. Here we have successfully constructed a micro in vitro environment with collagen based Extracellular Matrix (ECM) structures for cell 3-D metastasis. With stable nutrition (glucose) gradient inside, breast cancer cell MDA-MB-231 is able to invade inside the collagen from the nutrition poor site towards the nutrition rich site. Continuous confocal microscopy captures images of the cells every 12 hours and tracks their positions in 3-D space. The micro fluorescent beads pre-mixed inside the ECM demonstrate that invasive cells have altered the structures through mechanics. With the observation and the analysis of cell collective behaviors, we argue that game theory may exist between the pioneering cells and their followers in the metastatic cell group. The cell collaboration may explain the high efficiency of metastasis.

Cancer cell metabolism and the modulating effects of nitric oxide.

PubMed

Chang, Ching-Fang; Diers, Anne R; Hogg, Neil

2015-02-01

Altered metabolic phenotype has been recognized as a hallmark of tumor cells for many years, but this aspect of the cancer phenotype has come into greater focus in recent years. NOS2 (inducible nitric oxide synthase of iNOS) has been implicated as a component in many aggressive tumor phenotypes, including melanoma, glioblastoma, and breast cancer. Nitric oxide has been well established as a modulator of cellular bioenergetics pathways, in many ways similar to the alteration of cellular metabolism observed in aggressive tumors. In this review we attempt to bring these concepts together with the general hypothesis that one function of NOS2 and NO in cancer is to modulate metabolic processes to facilitate increased tumor aggression. There are many mechanisms by which NO can modulate tumor metabolism, including direct inhibition of respiration, alterations in mitochondrial mass, oxidative inhibition of bioenergetic enzymes, and the stimulation of secondary signaling pathways. Here we review metabolic alterations in the context of cancer cells and discuss the role of NO as a potential mediator of these changes. Copyright © 2015. Published by Elsevier Inc.

Cancer Cell Metabolism and the Modulating Effects of Nitric Oxide

PubMed Central

Chang, Ching-Fang; Diers, Anne R.; Hogg, Neil

2016-01-01

Altered metabolic phenotype has been recognized as a hallmark of tumor cells for many years, but this aspect of the cancer phenotype has come into greater focus in recent years. NOS2 (inducible nitric oxide synthase of iNOS) has been implicated as a component in many aggressive tumor phenotypes, including melanoma, glioblastoma and breast cancer. Nitric oxide has been well established as a modulator of cellular bioenergetics pathways, in many ways similar to the alteration of cellular metabolism observed in aggressive tumors. In this review we attempt to bring these concepts together with the general hypothesis that one function of NOS2 and NO in cancer is to modulate metabolic processes to facilitate increased tumor aggression. There are many mechanisms by which NO can modulate tumor metabolism, including direct inhibition of respiration, alterations in mitochondrial mass, oxidative inhibition of bioenergetic enzymes, and the stimulation of secondary signaling pathways. Here we review metabolic alterations in the context of cancer cells and discuss the role of NO as a potential mediator of these changes. PMID:25464273

Targeted Modification of Homogalacturonan by Transgenic Expression of a Fungal Polygalacturonase Alters Plant Growth1

PubMed Central

Capodicasa, Cristina; Vairo, Donatella; Zabotina, Olga; McCartney, Lesley; Caprari, Claudio; Mattei, Benedetta; Manfredini, Cinzia; Aracri, Benedetto; Benen, Jacques; Knox, J. Paul; De Lorenzo, Giulia; Cervone, Felice

2004-01-01

Pectins are a highly complex family of cell wall polysaccharides comprised of homogalacturonan (HGA), rhamnogalacturonan I and rhamnogalacturonan II. We have specifically modified HGA in both tobacco (Nicotiana tabacum) and Arabidopsis by expressing the endopolygalacturonase II of Aspergillus niger (AnPGII). Cell walls of transgenic tobacco plants showed a 25% reduction in GalUA content as compared with the wild type and a reduced content of deesterified HGA as detected by antibody labeling. Neutral sugars remained unchanged apart from a slight increase of Rha, Ara, and Gal. Both transgenic tobacco and Arabidopsis were dwarfed, indicating that unesterified HGA is a critical factor for plant cell growth. The dwarf phenotypes were associated with AnPGII activity as demonstrated by the observation that the mutant phenotype of tobacco was completely reverted by crossing the dwarfed plants with plants expressing PGIP2, a strong inhibitor of AnPGII. The mutant phenotype in Arabidopsis did not appear when transformation was performed with a gene encoding AnPGII inactivated by site directed mutagenesis. PMID:15247378

Analysis of antigen-specific B-cell memory directly ex vivo.

PubMed

McHeyzer-Williams, Louise J; McHeyzer-Williams, Michael G

2004-01-01

Helper T-cell-regulated B-cell memory develops in response to initial antigen priming as a cellular product of the germinal center (GC) reaction. On antigen recall, memory response precursors expand rapidly with exaggerated differentiation into plasma cells to produce the high-titer, high-affinity antibody(Ab) that typifies the memory B-cell response in vivo. We have devised a high-resolution flow cytometric strategy to quantify the emergence and maintenance of antigen-specific memory B cells directly ex vivo. Extended cell surface phenotype establishes a level of cellular diversity not previously appreciated for the memory B-cell compartment. Using an "exclusion transfer" strategy, we ascertain the capacity of two distinct memory B-cell populations to transfer antigen-specific memory into naive adoptive hosts. Finally, we sequence expressed messenger ribonucleic acid (mRNA) from single cells within the population to estimate the level of somatic hypermutation as the best molecular indicator of B-cell memory. In this chapter, we describe the methods used in each of these four sections that serve to provide high-resolution quantification of antigen-specific B-cell memory responses directly ex vivo.

Human fetal enterocytes in vitro: modulation of the phenotype by extracellular matrix.

PubMed Central

Sanderson, I R; Ezzell, R M; Kedinger, M; Erlanger, M; Xu, Z X; Pringault, E; Leon-Robine, S; Louvard, D; Walker, W A

1996-01-01

The differentiation of small intestinal epithelial cells may require stimulation by microenvironmental factors in vivo. In this study, the effects of mesenchymal and luminal elements in nonmalignant epithelia] cells isolated from the human fetus were studied in vitro. Enterocytes from the human fetus were cultured and microenvironmental factors were added in stages, each stage more closely approximating the microenvironment in vivo. Four stages were examined: epithelial cells derived on plastic from intestinal culture and grown as a cell clone, the same cells grown on connective tissue support, primary epithelial explants grown on fibroblasts with a laminin base, and primary epithelial explants grown on fibroblasts and laminin with n-butyrate added to the incubation medium. The epithelial cell clone dedifferentiated when grown on plastic; however, the cells expressed cytokeratins and villin as evidence of their epithelial cell origin. Human connective tissue matrix from Engelbreth-Holm-Swarm sarcoma cells (Matrigel) modulated their phenotype: alkaline phosphatase activity increased, microvilli developed on their apical surface, and the profile of insulin-like growth factor binding proteins resembled that secreted by differentiated enterocytes. Epithelial cells taken directly from the human fetus as primary cultures and grown as explants on fibroblasts and laminin expressed greater specific enzyme activities in brush border membrane fractions than the cell clone. These activities were enhanced by the luminal molecule sodium butyrate. Thus the sequential addition of connective tissue and luminal molecules to nonmalignant epithelia] cells in vitro induces a spectrum of changes in the epithelial cell phenotype toward full differentiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8755542

Bi-directional exchange of membrane components occurs during co-culture of mesenchymal stem cells and nucleus pulposus cells.

PubMed

Strassburg, Sandra; Hodson, Nigel W; Hill, Patrick I; Richardson, Stephen M; Hoyland, Judith A

2012-01-01

Mesenchymal stem cell (MSC)-based therapies have been proposed as novel treatments for intervertebral disc (IVD) degeneration. We have previously demonstrated that when MSCs are co-cultured with nucleus pulposus (NP) cells with direct cell-cell contact, they differentiate along the NP lineage and simultaneously stimulate the degenerate NP cell population to regain a normal (non-degenerate) phenotype, an effect which requires cell-cell communication. However, the mechanisms by which NP cells and MSCs interact in this system are currently unclear. Thus, in this study we investigated a range of potential mechanisms for exchange of cellular components or information that may direct these changes, including cell fusion, gap-junctional communication and exchange of membrane components by direct transfer or via microvesicle formation. Flow cytometry of fluorescently labeled MSCs and NP cells revealed evidence of some cell fusion and formation of gapjunctions, although at the three timepoints studied these phenomena were detectable only in a small proportion of cells. While these mechanisms may play a role in cell-cell communication, the data suggests they are not the predominant mechanism of interaction. However, flow cytometry of fluorescently dual-labeled cells showed that extensive bi-directional transfer of membrane components is operational during direct co-culture of MSCs and NP cells. Furthermore, there was also evidence for secretion and internalization of membrane-bound microvesicles by both cell types. Thus, this study highlights bi-directional intercellular transfer of membrane components as a possible mechanism of cellular communication between MSC and NP cells.

miR-300 inhibits epithelial to mesenchymal transition and metastasis by targeting Twist in human epithelial cancer

PubMed Central

2014-01-01

Background Epithelial-to-mesenchymal transition (EMT) is a key step of the progression of tumor cell metastasis. Recent work has demonstrated some miRNAs play critical roles in EMT. In this study, we focused on the roles of miR-300 in regulating EMT. Methods The expression levels of miR-300 were examined in epithelial carcinoma cells that underwent an EMT using quantitative reverse transcription-PCR. The role of miR-300 in EMT was investigated by transfection of the miR-300 mimic or inhibitor in natural epithelial-mesenchymal phenotype cell line pairs and in transforming growth factor (TGF) beta-induced EMT cell models. A luciferase reporter assay and a rescue experiment were conducted to confirm the target gene of miR-300. The efficacy of miR-300 against tumor invasion and metastasis was evaluated both in vitro and in vivo. Correlation analysis between miR-300 expression and the expression levels of its target gene, as well as tumor metastasis was performed in specimens from patients with head and neck squamous cell carcinoma (HNSCC). Results MiR-300 was found down-regulated in the HNSCC cells and breast cancer cells that underwent EMT. Ectopic expression of miR-300 effectively blocked TGF-beta-induced EMT and reversed the phenotype of EMT in HN-12 and MDA-MB-231 cells, but inhibition of miR-300 in the epithelial phenotype cells, HN-4 and MCF-7 cells, could induce EMT. The luciferase reporter assay and the rescue assay results showed that miR-300 directly targets the 3′UTR of Twist. Enforced miR-300 expression suppressed cell invasion in vitro and experimental metastasis in vivo. Clinically, miR-300 expression was found inversely correlated with Twist expression and reduced miR-300 was associated with metastasis in patient specimens. Conclusions Down-regulation of miR-300 is required for EMT initiation and maintenance. MiR-300 may negatively regulate EMT by direct targeting Twist and therefore inhibit cancer cell invasion and metastasis, which implicates miR-300 as an attractive candidate for cancer therapy. PMID:24885626

miR-300 inhibits epithelial to mesenchymal transition and metastasis by targeting Twist in human epithelial cancer.

PubMed

Yu, Jingshuang; Xie, Furong; Bao, Xin; Chen, Wantao; Xu, Qin

2014-05-24

Epithelial-to-mesenchymal transition (EMT) is a key step of the progression of tumor cell metastasis. Recent work has demonstrated some miRNAs play critical roles in EMT. In this study, we focused on the roles of miR-300 in regulating EMT. The expression levels of miR-300 were examined in epithelial carcinoma cells that underwent an EMT using quantitative reverse transcription-PCR. The role of miR-300 in EMT was investigated by transfection of the miR-300 mimic or inhibitor in natural epithelial-mesenchymal phenotype cell line pairs and in transforming growth factor (TGF) beta-induced EMT cell models. A luciferase reporter assay and a rescue experiment were conducted to confirm the target gene of miR-300. The efficacy of miR-300 against tumor invasion and metastasis was evaluated both in vitro and in vivo. Correlation analysis between miR-300 expression and the expression levels of its target gene, as well as tumor metastasis was performed in specimens from patients with head and neck squamous cell carcinoma (HNSCC). MiR-300 was found down-regulated in the HNSCC cells and breast cancer cells that underwent EMT. Ectopic expression of miR-300 effectively blocked TGF-beta-induced EMT and reversed the phenotype of EMT in HN-12 and MDA-MB-231 cells, but inhibition of miR-300 in the epithelial phenotype cells, HN-4 and MCF-7 cells, could induce EMT. The luciferase reporter assay and the rescue assay results showed that miR-300 directly targets the 3'UTR of Twist. Enforced miR-300 expression suppressed cell invasion in vitro and experimental metastasis in vivo. Clinically, miR-300 expression was found inversely correlated with Twist expression and reduced miR-300 was associated with metastasis in patient specimens. Down-regulation of miR-300 is required for EMT initiation and maintenance. MiR-300 may negatively regulate EMT by direct targeting Twist and therefore inhibit cancer cell invasion and metastasis, which implicates miR-300 as an attractive candidate for cancer therapy.

The Twist Box Domain is Required for Twist1-induced Prostate Cancer Metastasis

PubMed Central

Gajula, Rajendra P.; Chettiar, Sivarajan T.; Williams, Russell D.; Thiyagarajan, Saravanan; Kato, Yoshinori; Aziz, Khaled; Wang, Ruoqi; Gandhi, Nishant; Wild, Aaron T.; Vesuna, Farhad; Ma, Jinfang; Salih, Tarek; Cades, Jessica; Fertig, Elana; Biswal, Shyam; Burns, Timothy F.; Chung, Christine H.; Rudin, Charles M.; Herman, Joseph M.; Hales, Russell K.; Raman, Venu; An, Steven S.; Tran, Phuoc T.

2013-01-01

Twist1, a basic helix-loop-helix transcription factor, plays a key role during development and is a master regulator of the epithelial-mesenchymal transition (EMT) that promotes cancer metastasis. Structure-function relationships of Twist1 to cancer-related phenotypes are underappreciated, so we studied the requirement of the conserved Twist box domain for metastatic phenotypes in prostate cancer (PCa). Evidence suggests that Twist1 is overexpressed in clinical specimens and correlated with aggressive/metastatic disease. Therefore, we examined a transactivation mutant, Twist1-F191G, in PCa cells using in vitro assays which mimic various stages of metastasis. Twist1 overexpression led to elevated cytoskeletal stiffness and cell traction forces at the migratory edge of cells based on biophysical single-cell measurements. Twist1 conferred additional cellular properties associated with cancer cell metastasis including increased migration, invasion, anoikis resistance, and anchorage-independent growth. The Twist box mutant was defective for these Twist1 phenotypes in vitro. Importantly, we observed a high frequency of Twist1-induced metastatic lung tumors and extra-thoracic metastases in vivo using the experimental lung metastasis assay. The Twist box was required for PCa cells to colonize metastatic lung lesions and extra-thoracic metastases. Comparative genomic profiling revealed transcriptional programs directed by the Twist box that were associated with cancer progression, such as Hoxa9. Mechanistically, Twist1 bound to the Hoxa9 promoter and positively regulated Hoxa9 expression in PCa cells. Finally, Hoxa9 was important for Twist1-induced cellular phenotypes associated with metastasis. These data suggest that the Twist box domain is required for Twist1 transcriptional programs and PCa metastasis. PMID:23982216

Interactions between peripheral blood CD8 T lymphocytes and intestinal epithelial cells (iEC).

PubMed

Arosa, F A; Irwin, C; Mayer, L; de Sousa, M; Posnett, D N

1998-05-01

Intestinal intraepithelial lymphocytes (iIEL) are primarily CD8 cells and most of them have a CD28- phenotype, the phenotype of effector cytotoxic T cells. We asked whether the predominance of CD8+CD28- T cells in the gut may result from peripheral blood T cells preferentially migrating to the iIEL compartment and adhering to iEC. Compared with CD4 cells, adhesion of resting CD8+ T cells to iEC cell lines was significantly higher. Adhesion could be blocked with a MoAb to gp180, a molecule expressed on iEC which is known to interact with CD8/lck. No significant difference in the level of adhesion was observed between CD8+CD28+ and CD8+CD28- T cells. Thus CD8 cells may preferentially migrate to the iIEL compartment, but loss of CD28 expression could occur in situ after migration. Consistent with this hypothesis, the CD8+CD28- cells became enriched after co-culturing T cells with iEC cell lines and primary iEC. Induction of the CD8+CD28- phenotype in cord blood and adult T cells was observed in co-cultures with iEC and also with mitogens and superantigens. In the latter case, CD28 down-modulation was seen specifically in the Vbeta subset targeted by the superantigen, indicating that loss of CD28 expression is a direct result of T cell receptor (TCR)-mediated stimulation. The combined results suggest that CD8+CD28- T cells are antigen experienced T cells, and that they may have a survival advantage in the presence of gut epithelial cells in vitro. This may contribute to the predominance of CD8+CD28- T cells in the iIEL compartment.

S100A8/A9 regulates MMP-2 expression and invasion and migration by carcinoma cells

PubMed Central

Silva, Emmanuel J.; Argyris, Prokopios P.; Zou, Xianqiong; Ross, Karen F.; Herzberg, Mark C.

2014-01-01

Intracellular calprotectin (S100A8/A9) functions in the control of the cell cycle checkpoint at G2/M. Dysregulation of S100A8/A9 appears to cause loss of the checkpoint, which frequently characterizes head and neck squamous cell carcinoma (HNSCC). In the present study, we analyzed carcinoma cells for other S100A8/A9-directed changes in malignant phenotype. Using a S100A8/A9-negative human carcinoma cell line (KB), transfection to express S100A8 and S100A9 caused selective down-regulation of MMP-2 and inhibited in vitro invasion and migration. Conversely, silencing of endogenous S100A8 and S100A9 expression in TR146 cells, a well-differentiated HNSCC cell line, increased MMP-2 activity and in vitro invasion and migration. When MMP-2 expression was silenced, cells appeared to assume a less malignant phenotype. To more closely model the architecture of cell growth in vivo, cells were grown in a 3D collagen substrate, which was compared to 2D. Growth on 3D substrates caused greater MMP-2 expression. Whereas hypermethylation of CpG islands occurs frequently in HNSCC, S100A8/A9-dependent regulation of MMP-2 could not be explained by modification of the upstream promoters of MMP2 or TIMP2. Collectively, these results suggest that intracellular S100A8/A9 contributes to the cancer cell phenotype by modulating MMP-2 expression and activity to regulate cell migration and mobility. PMID:25236491

p21(WAF1) Mediates Cell-Cycle Inhibition, Relevant to Cancer Suppression and Therapy.

PubMed

El-Deiry, Wafik S

2016-09-15

p21 (WAF1/CIP1; CDKN1a) is a universal cell-cycle inhibitor directly controlled by p53 and p53-independent pathways. Knowledge of the regulation and function of p21 in normal and cancer cells has opened up several areas of investigation and has led to novel therapeutic strategies. The discovery in 1993 and subsequent work on p21 has illuminated basic cellular growth control, stem cell phenotypes, the physiology of differentiation, as well as how cells respond to stress. There remain open questions in the signaling networks, the ultimate role of p21 in the p53-deficiency phenotype in the context of other p53 target defects, and therapeutic strategies continue to be a work in progress. Cancer Res; 76(18); 5189-91. ©2016 AACRSee related article by El-Deiry et al., Cancer Res 1994;54:1169-74Visit the Cancer Research 75(th) Anniversary timeline. ©2016 American Association for Cancer Research.

Direct testing for allele-specific expression differences between conditions

USDA-ARS?s Scientific Manuscript database

Genetic differences in cis regulatory regions contribute to the phenotypic variation observed in natural and human populations, including beneficial, potentially adaptive, traits as well as disease states. The two alleles in a diploid cell can differ in their allele-specific expression leading to al...

Does the central dogma still stand?

PubMed Central

2012-01-01

Abstract Prions are agents of analog, protein conformation-based inheritance that can confer beneficial phenotypes to cells, especially under stress. Combined with genetic variation, prion-mediated inheritance can be channeled into prion-independent genomic inheritance. Latest screening shows that prions are common, at least in fungi. Thus, there is non-negligible flow of information from proteins to the genome in modern cells, in a direct violation of the Central Dogma of molecular biology. The prion-mediated heredity that violates the Central Dogma appears to be a specific, most radical manifestation of the widespread assimilation of protein (epigenetic) variation into genetic variation. The epigenetic variation precedes and facilitates genetic adaptation through a general ‘look-ahead effect’ of phenotypic mutations. This direction of the information flow is likely to be one of the important routes of environment-genome interaction and could substantially contribute to the evolution of complex adaptive traits. Reviewers This article was reviewed by Jerzy Jurka, Pierre Pontarotti and Juergen Brosius. For the complete reviews, see the Reviewers’ Reports section. PMID:22913395

A targeted genetic modifier screen links the SWI2/SNF2 protein domino to growth and autophagy genes in Drosophila melanogaster.

PubMed

Kwon, Matt Hyoung; Callaway, Heather; Zhong, Jim; Yedvobnick, Barry

2013-05-20

Targeted genetic studies can facilitate phenotypic analyses and provide important insights into development and other complex processes. The SWI2/SNF2 DNA-dependent ATPase Domino (Dom) of Drosophila melanogaster, a component of the Tip60 acetyltransferase complex, has been associated with a wide spectrum of cellular processes at multiple developmental stages. These include hematopoiesis, cell proliferation, homeotic gene regulation, histone exchange during DNA repair, and Notch signaling. To explore the wider gene network associated with Dom action, we used RNAi directed against domino (dom) to mediate loss-of-function at the wing margin, a tissue that is readily scored for phenotypic changes. Dom RNAi driven through GAL4-UAS elicited dominant wing nicking that responded phenotypically to the dose of dom and other loci known to function with dom. We screened for phenotypic modifiers of this wing phenotype among 2500 transpositions of the EP P element and found both enhancers and suppressors. Several classes of modifier were obtained, including those encoding transcription factors, RNA regulatory proteins, and factors that regulate cell growth, proliferation and autophagy, a lysosomal degradation pathway that affects cell growth under conditions of starvation and stress. Our analysis is consistent with prior studies, suggesting that Dom acts pleiotropically as a positive effector of Notch signaling and a repressor of proliferation. This genetic system should facilitate screens for additional loci associated with Dom function, and complement biochemical approaches to their regulatory activity.

Human Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells in Phenotypic Screening: A Transforming Growth Factor-β Type 1 Receptor Kinase Inhibitor Induces Efficient Cardiac Differentiation.

PubMed

Drowley, Lauren; Koonce, Chad; Peel, Samantha; Jonebring, Anna; Plowright, Alleyn T; Kattman, Steven J; Andersson, Henrik; Anson, Blake; Swanson, Bradley J; Wang, Qing-Dong; Brolen, Gabriella

2016-02-01

Several progenitor cell populations have been reported to exist in hearts that play a role in cardiac turnover and/or repair. Despite the presence of cardiac stem and progenitor cells within the myocardium, functional repair of the heart after injury is inadequate. Identification of the signaling pathways involved in the expansion and differentiation of cardiac progenitor cells (CPCs) will broaden insight into the fundamental mechanisms playing a role in cardiac homeostasis and disease and might provide strategies for in vivo regenerative therapies. To understand and exploit cardiac ontogeny for drug discovery efforts, we developed an in vitro human induced pluripotent stem cell-derived CPC model system using a highly enriched population of KDR(pos)/CKIT(neg)/NKX2.5(pos) CPCs. Using this model system, these CPCs were capable of generating highly enriched cultures of cardiomyocytes under directed differentiation conditions. In order to facilitate the identification of pathways and targets involved in proliferation and differentiation of resident CPCs, we developed phenotypic screening assays. Screening paradigms for therapeutic applications require a robust, scalable, and consistent methodology. In the present study, we have demonstrated the suitability of these cells for medium to high-throughput screens to assess both proliferation and multilineage differentiation. Using this CPC model system and a small directed compound set, we identified activin-like kinase 5 (transforming growth factor-β type 1 receptor kinase) inhibitors as novel and potent inducers of human CPC differentiation to cardiomyocytes. Significance: Cardiac disease is a leading cause of morbidity and mortality, with no treatment available that can result in functional repair. This study demonstrates how differentiation of induced pluripotent stem cells can be used to identify and isolate cell populations of interest that can translate to the adult human heart. Two separate examples of phenotypic screens are discussed, demonstrating the value of this biologically relevant and reproducible technology. In addition, this assay system was able to identify novel and potent inducers of differentiation and proliferation of induced pluripotent stem cell-derived cardiac progenitor cells. ©AlphaMed Press.

Characterization of mesenchymal stem cells and fibrochondrocytes in three-dimensional co-culture: analysis of cell shape, matrix production, and mechanical performance.

PubMed

McCorry, Mary Clare; Puetzer, Jennifer L; Bonassar, Lawrence J

2016-03-12

Bone marrow mesenchymal stem cells (MSCs) have shown positive therapeutic effects for meniscus regeneration and repair. Preliminary in vitro work has indicated positive results for MSC applications for meniscus tissue engineering; however, more information is needed on how to direct MSC behavior. The objective of this study was to examine the effect of MSC co-culture with primary meniscal fibrochondrocytes (FCCs) in a three-dimensional collagen scaffold in fibrochondrogenic media. Co-culture of MSCs and FCCs was hypothesized to facilitate the transition of MSCs to a FCC cell phenotype as measured by matrix secretion and morphology. MSCs and FCCs were isolated from bovine bone marrow and meniscus, respectively. Cells were seeded in a 20 mg/mL high-density type I collagen gel at MSC:FCC ratios of 0:100, 25:75, 50:50, 75:25, and 100:0. Constructs were cultured for up to 2 weeks and then analyzed for cell morphology, glycosaminoglycan content, collagen content, and production of collagen type I, II, and X. Cells were homogeneously mixed throughout the scaffold and cells had limited direct cell-cell contact. After 2 weeks in culture, MSCs transitioned from a spindle-like morphology toward a rounded phenotype, while FCCs remained rounded throughout culture. Although MSC shape changed with culture, the overall size was significantly larger than FCCs throughout culture. While 75:25 and 100:0 (MSC mono-culture) culture groups produced significantly more glycosaminoglycan (GAG)/DNA than FCCs in mono-culture, GAG retention was highest in 50:50 co-cultures. Similarly, the aggregate modulus was highest in 100:0 and 50:50 co-cultures. All samples contained both collagen types I and II after 2 weeks, and collagen type X expression was evident only in MSC mono-culture gels. MSCs shift to a FCC morphology in both mono- and co-culture. Co-culture reduced hypertrophy by MSCs, indicated by collagen type X. This study shows that MSC phenotype can be influenced by indirect homogeneous cell culture in a three-dimensional gel, demonstrating the applicability of MSCs in meniscus tissue engineering applications.

The flipflop orphan genes are required for limb bud eversion in the Tribolium embryo.

PubMed

Thümecke, Susanne; Beermann, Anke; Klingler, Martin; Schröder, Reinhard

2017-01-01

Unlike Drosophila but similar to other arthropod and vertebrate embryos, the flour beetle Tribolium castaneum develops everted limb buds during embryogenesis. However, the molecular processes directing the evagination of epithelia are only poorly understood. Here we show that the newly discovered genes Tc-flipflop1 and Tc-flipflop2 are involved in regulating the directional budding of appendages. RNAi-knockdown of Tc-flipflop results in a variety of phenotypic traits. Most prominently, embryonic limb buds frequently grow inwards rather than out, leading to the development of inverted appendages inside the larval body. Moreover, affected embryos display dorsal closure defects. The Tc-flipflop genes are evolutionarily non-conserved, and their molecular function is not evident. We further found that Tc-RhoGEF2 , a highly-conserved gene known to be involved in actomyosin-dependent cell movement and cell shape changes, shows a Tc-flipflop -like RNAi-phenotype. The similarity of the inverted appendage phenotype in both the flipflop - and the RhoGEF2 RNAi gene knockdown led us to conclude that the Tc-flipflop orphan genes act in a Rho-dependent pathway that is essential for the early morphogenesis of polarised epithelial movements. Our work describes one of the few examples of an orphan gene playing a crucial role in an important developmental process.

Programmed Death 1 Regulates Memory Phenotype CD4 T Cell Accumulation, Inhibits Expansion of the Effector Memory Phenotype Subset and Modulates Production of Effector Cytokines

PubMed Central

Charlton, Joanna J.; Tsoukatou, Debbie; Mamalaki, Clio; Chatzidakis, Ioannis

2015-01-01

Memory phenotype CD4 T cells are found in normal mice and arise through response to environmental antigens or homeostatic mechanisms. The factors that regulate the homeostasis of memory phenotype CD4 cells are not clear. In the present study we demonstrate that there is a marked accumulation of memory phenotype CD4 cells, specifically of the effector memory (TEM) phenotype, in lymphoid organs and tissues of mice deficient for the negative co-stimulatory receptor programmed death 1 (PD-1). This can be correlated with decreased apoptosis but not with enhanced homeostatic turnover potential of these cells. PD-1 ablation increased the frequency of memory phenotype CD4 IFN-γ producers but decreased the respective frequency of IL-17A-producing cells. In particular, IFN-γ producers were more abundant but IL-17A producing cells were more scarce among PD-1 KO TEM-phenotype cells relative to WT. Transfer of peripheral naïve CD4 T cells suggested that accumulated PD-1 KO TEM-phenotype cells are of peripheral and not of thymic origin. This accumulation effect was mediated by CD4 cell-intrinsic mechanisms as shown by mixed bone marrow chimera experiments. Naïve PD-1 KO CD4 T cells gave rise to higher numbers of TEM-phenotype lymphopenia-induced proliferation memory cells. In conclusion, we provide evidence that PD-1 has an important role in determining the composition and functional aspects of memory phenotype CD4 T cell pool. PMID:25803808

Concise review: reprogramming strategies for cardiovascular regenerative medicine: from induced pluripotent stem cells to direct reprogramming.

PubMed

Budniatzky, Inbar; Gepstein, Lior

2014-04-01

Myocardial cell-replacement therapies are emerging as novel therapeutic paradigms for myocardial repair but are hampered by the lack of sources of autologous human cardiomyocytes. The recent advances in stem cell biology and in transcription factor-based reprogramming strategies may provide exciting solutions to this problem. In the current review, we describe the different reprogramming strategies that can give rise to cardiomyocytes for regenerative medicine purposes. Initially, we describe induced pluripotent stem cell technology, a method by which adult somatic cells can be reprogrammed to yield pluripotent stem cells that could later be coaxed ex vivo to differentiate into cardiomyocytes. The generated induced pluripotent stem cell-derived cardiomyocytes could then be used for myocardial cell transplantation and tissue engineering strategies. We also describe the more recent direct reprogramming approaches that aim to directly convert the phenotype of one mature cell type (fibroblast) to another (cardiomyocyte) without going through a pluripotent intermediate cell type. The advantages and shortcomings of each strategy for cardiac regeneration are discussed, along with the hurdles that need to be overcome on the road to clinical translation.

Mast Cell Function

PubMed Central

da Silva, Elaine Zayas Marcelino; Jamur, Maria Célia

2014-01-01

Since first described by Paul Ehrlich in 1878, mast cells have been mostly viewed as effectors of allergy. It has been only in the past two decades that mast cells have gained recognition for their involvement in other physiological and pathological processes. Mast cells have a widespread distribution and are found predominantly at the interface between the host and the external environment. Mast cell maturation, phenotype and function are a direct consequence of the local microenvironment and have a marked influence on their ability to specifically recognize and respond to various stimuli through the release of an array of biologically active mediators. These features enable mast cells to act as both first responders in harmful situations as well as to respond to changes in their environment by communicating with a variety of other cells implicated in physiological and immunological responses. Therefore, the critical role of mast cells in both innate and adaptive immunity, including immune tolerance, has gained increased prominence. Conversely, mast cell dysfunction has pointed to these cells as the main offenders in several chronic allergic/inflammatory disorders, cancer and autoimmune diseases. This review summarizes the current knowledge of mast cell function in both normal and pathological conditions with regards to their regulation, phenotype and role. PMID:25062998

RB Loss Promotes Prostate Cancer Metastasis

PubMed Central

Thangavel, Chellappagounder; Boopathi, Ettickan; Liu, Yi; Haber, Alex; Ertel, Adam; Bhardwaj, Anshul; Addya, Sankar; Williams, Noelle; Ciment, Stephen J.; Cotzia, Paolo; Dean, Jeffry L.; Snook, Adam; McNair, Chris; Price, Matt; Hernandez, James R.; Zhao, Shuang G.; Birbe, Ruth; McCarthy, James B.; Turley, Eva A.; Pienta, Kenneth J.; Feng, Felix Y.; Dicker, Adam P.; Knudsen, Karen E.; Den, Robert B.

2017-01-01

RB loss occurs commonly in neoplasia but its contributions to advanced cancer have not been assessed directly. Here we show that RB loss in multiple murine models of cancer produces a prometastatic phenotype. Gene expression analyses showed that regulation of the cell motility receptor RHAMM by the RB/E2F pathway was critical for epithelial–mesenchymal transition, motility, and invasion by cancer cells. Genetic modulation or pharmacologic inhibition of RHAMM activity was sufficient and necessary for metastatic phenotypes induced by RB loss in prostate cancer. Mechanistic studies in this setting established that RHAMM stabilized F-actin polymerization by controlling ROCK signaling. Collectively, our findings show how RB loss drives metastatic capacity and highlight RHAMM as a candidate therapeutic target for treating advanced prostate cancer. PMID:27923835

Chemical Fluxes in Cellular Steady States Measured by Fluorescence Correlation Spectroscopy

NASA Astrophysics Data System (ADS)

Qian, Hong; Elson, Elliot L.

Genetically, identical cells adopt phenotypes that have different structures, functions, and metabolic properties. In multi-cellular organisms, for example, tissue-specific phenotypes distinguish muscle cells, liver cells, fibroblasts, and blood cells that differ in biochemical functions, geometric forms, and interactions with extracellular environments. Tissue-specific cells usually have different metabolic functions such as synthesis of distinct spectra of secreted proteins, e.g., by liver or pancreatic cells, or of structural proteins, e.g., muscle vs. epithelial cells. But more importantly, a phenotype should include a dynamic aspect: different phenotypes can have distinctly different dynamic functions such as contraction of muscle cells and locomotion of leukocytes. The phenotypes of differentiated tissue cells are typically stable, but they can respond to changes in external conditions, e.g., as in the hypertrophy of muscle cells in response to extra load [1] or the phenotypic shift of fibroblasts to myofibroblasts as part of the wound healing response [2]. Cells pass through sequences of phenotypes during development and also undergo malignant phenotypic transformations as occur in cancer and heart disease.

Col1A1 Production and Apoptotic Resistance in TGF-β1-Induced Epithelial-to-Mesenchymal Transition-Like Phenotype of 603B Cells

PubMed Central

Liu, Jun; Eischeid, Alex N.; Chen, Xian-Ming

2012-01-01

Recent studies have suggested that proliferating cholangiocytes have an important role in the induction of fibrosis, either directly via epithelial-to-mesenchymal transition (EMT), or indirectly via activation of other liver cell types. Transforming growth factor beta 1 (TGF-β1), a critical fibrotic cytokine for hepatic fibrosis, is a potent EMT inducer. This study aimed to clarify the potential contributions of TGF-β1-induced EMT-like cholangiocyte phenotype to collagen production and cell survival of cholangiocytes in vitro. Mouse cholangiocytes (603B cells) were treated with TGF-β1 and EMT-like phenotype alterations were monitored by morphological changes and expression of EMT-associated genes. Alterations in Col1A1 gene, Col1A1-associated miR-29s, and pro-apoptotic genes were measured in TGF-β1-treated 603B cells. Snail1 knockdown was achieved using shRNA to evaluate the contribution of EMT-associated changes to Col1A1 production and cell survival. We found TGF-β1 treatment induced partial EMT-like phenotype transition in 603B cells in a Snail1-dependent manner. TGF-β1 also stimulated collagen α1(I) expression in 603B cells. However, this induction was not parallel to the EMT-like alterations and independent of Snail1 or miR-29 expression. Cells undergoing EMT-like changes showed a modest down-regulation of multiple pro-apoptotic genes and displayed resistance to TNF-α-induced apoptosis. TGF-β1-induced apoptosis resistance was attenuated in Snail1 knockdown 603B cells. TGF-β1-induced Col1A1 production seems to be independent of EMT-like transition and miR-29 expression. Nevertheless, TGF-β1-induced EMT may contribute to the increased survival capacity of cholangiocytes via modulating the expression of pro-apoptotic genes. PMID:23236489

Correlation of RNA secondary structure and attenuation of Sabin vaccine strains of poliovirus in tissue culture.

PubMed

Macadam, A J; Ferguson, G; Burlison, J; Stone, D; Skuce, R; Almond, J W; Minor, P D

1992-08-01

Part of the 5' noncoding regions of all three Sabin vaccine strains of poliovirus contains determinants of attenuation that are shown here to influence the ability of these strains to grow at elevated temperatures in BGM cells. The predicted RNA secondary structure of this region (nt 464-542 in P3/Sabin) suggests that both phenotypes are due to perturbation of base-paired stems. Ts phenotypes of site-directed mutants with defined changes in this region correlated well with predicted secondary structure stabilities. Reversal of base-pair orientation had little effect whereas stem disruption led to marked increases in temperature sensitivity. Phenotypic revertants of such viruses displayed mutations on either side of the stem. Mutations destabilizing stems led to intermediate phenotypes. These results provided evidence for the biological significance of the predicted RNA secondary structure.

Dual gene activation and knockout screen reveals directional dependencies in genetic networks. | Office of Cancer Genomics

Cancer.gov

Understanding the direction of information flow is essential for characterizing how genetic networks affect phenotypes. However, methods to find genetic interactions largely fail to reveal directional dependencies. We combine two orthogonal Cas9 proteins from Streptococcus pyogenes and Staphylococcus aureus to carry out a dual screen in which one gene is activated while a second gene is deleted in the same cell. We analyze the quantitative effects of activation and knockout to calculate genetic interaction and directionality scores for each gene pair.

Transition of late-stage effector T cells to CD27+ CD28+ tumor-reactive effector memory T cells in humans after adoptive cell transfer therapy

PubMed Central

Powell, Daniel J.; Dudley, Mark E.; Robbins, Paul F.; Rosenberg, Steven A.

2007-01-01

In humans, the pathways of memory T-cell differentiation remain poorly defined. Recently, adoptive cell transfer (ACT) of tumor-reactive T lymphocytes to metastatic melanoma patients after nonmyeloablative chemotherapy has resulted in persistence of functional, tumor-reactive lymphocytes, regression of disease, and induction of melanocyte-directed autoimmunity in some responding patients. In the current study, longitudinal phenotypic analysis was performed on melanoma antigen–specific CD8+ T cells during their transition from in vitro cultured effector cells to long-term persistent memory cells following ACT to 6 responding patients. Tumor-reactive T cells used for therapy were generally late-stage effector cells with a CD27Lo CD28Lo CD45RA− CD62 ligand− (CD62L−) CC chemokine receptor 7− (CCR7−) interleukin-7 receptor αLo (IL-7RαLo) phenotype. After transfer, rapid up-regulation and continued expression of IL-7Rα in vivo suggested an important role for IL-7R in immediate and long-term T-cell survival. Although the tumor antigen–specific T-cell population contracted between 1 and 4 weeks after transfer, stable numbers of CD27+ CD28+ tumor-reactive T cells were maintained, demonstrating their contribution to the development of long-term, melanoma-reactive memory CD8+ T cells in vivo. At 2 months after transfer, melanoma-reactive T cells persisted at high levels and displayed an effector memory phenotype, including a CD27+ CD28+ CD62L− CCR7− profile, which may explain in part their ability to mediate tumor destruction. PMID:15345595

Phenotypic and genotypic analysis of Borrelia burgdorferi isolates from various sources.

PubMed Central

Adam, T; Gassmann, G S; Rasiah, C; Göbel, U B

1991-01-01

A total of 17 B. burgdorferi isolates from various sources were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, restriction enzyme analysis, Southern hybridization with probes complementary to unique regions of evolutionarily conserved genes (16S rRNA and fla), and direct sequencing of in vitro polymerase chain reaction-amplified fragments of the 16S rRNA gene. Three groups were distinguished on the basis of phenotypic and genotypic traits, the latter traced to the nucleotide sequence level. Images PMID:1649797

GBM heterogeneity as a function of variable epidermal growth factor receptor variant III activity.

PubMed

Lindberg, Olle R; McKinney, Andrew; Engler, Jane R; Koshkakaryan, Gayane; Gong, Henry; Robinson, Aaron E; Ewald, Andrew J; Huillard, Emmanuelle; David James, C; Molinaro, Annette M; Shieh, Joseph T; Phillips, Joanna J

2016-11-29

Abnormal activation of the epidermal growth factor receptor (EGFR) due to a deletion of exons 2-7 of EGFR (EGFRvIII) is a common alteration in glioblastoma (GBM). While this alteration can drive gliomagenesis, tumors harboring EGFRvIII are heterogeneous. To investigate the role for EGFRvIII activation in tumor phenotype we used a neural progenitor cell-based murine model of GBM driven by EGFR signaling and generated tumor progenitor cells with high and low EGFRvIII activation, pEGFRHi and pEGFRLo. In vivo, ex vivo, and in vitro studies suggested a direct association between EGFRvIII activity and increased tumor cell proliferation, decreased tumor cell adhesion to the extracellular matrix, and altered progenitor cell phenotype. Time-lapse confocal imaging of tumor cells in brain slice cultures demonstrated blood vessel co-option by tumor cells and highlighted differences in invasive pattern. Inhibition of EGFR signaling in pEGFRHi promoted cell differentiation and increased cell-matrix adhesion. Conversely, increased EGFRvIII activation in pEGFRLo reduced cell-matrix adhesion. Our study using a murine model for GBM driven by a single genetic driver, suggests differences in EGFR activation contribute to tumor heterogeneity and aggressiveness.

True-breeding targeted gene knock-out in barley using designer TALE-nuclease in haploid cells.

PubMed

Gurushidze, Maia; Hensel, Goetz; Hiekel, Stefan; Schedel, Sindy; Valkov, Vladimir; Kumlehn, Jochen

2014-01-01

Transcription activator-like effector nucleases (TALENs) are customizable fusion proteins able to cleave virtually any genomic DNA sequence of choice, and thereby to generate site-directed genetic modifications in a wide range of cells and organisms. In the present study, we expressed TALENs in pollen-derived, regenerable cells to establish the generation of instantly true-breeding mutant plants. A gfp-specific TALEN pair was expressed via Agrobacterium-mediated transformation in embryogenic pollen of transgenic barley harboring a functional copy of gfp. Thanks to the haploid nature of the target cells, knock-out mutations were readily detected, and homozygous primary mutant plants obtained following genome duplication. In all, 22% of the TALEN transgenics proved knocked out with respect to gfp, and the loss of function could be ascribed to the deletions of between four and 36 nucleotides in length. The altered gfp alleles were transmitted normally through meiosis, and the knock-out phenotype was consistently shown by the offspring of two independent mutants. Thus, here we describe the efficient production of TALEN-mediated gene knock-outs in barley that are instantaneously homozygous and non-chimeric in regard to the site-directed mutations induced. This TALEN approach has broad applicability for both elucidating gene function and tailoring the phenotype of barley and other crop species.

Coculture with endothelial cells reduces the population of cycling LeX neural precursors but increases that of quiescent cells with a side population phenotype

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mathieu, Celine; Fouchet, Pierre; Gauthier, Laurent R.

2006-04-01

Neural stem cell proliferation and differentiation are regulated by external cues from their microenvironment. As endothelial cells are closely associated with neural stem cell in brain germinal zones, we investigated whether endothelial cells may interfere with neurogenesis. Neural precursor cells (NPC) from telencephalon of EGFP mouse embryos were cocultured in direct contact with endothelial cells. Endothelial cells did not modify the overall proliferation and apoptosis of neural cells, albeit they transiently delayed spontaneous apoptosis. These effects appeared to be specific to endothelial cells since a decrease in proliferation and a raise in apoptosis were observed in cocultures with fibroblasts. Endothelialmore » cells stimulated the differentiation of NPC into astrocytes and into neurons, whereas they reduced differentiation into oligodendrocytes in comparison to adherent cultures on polyornithine. Determination of NPC clonogenicity and quantification of LeX expression, a marker for NPC, showed that endothelial cells decreased the number of cycling NPC. On the other hand, the presence of endothelial cells increased the number of neural cells having 'side population' phenotype, another marker reported on NPC, which we have shown to contain quiescent cells. Thus, we show that endothelial cells may regulate neurogenesis by acting at different level of NPC differentiation, proliferation and quiescence.« less

Distinct CD4+CD8+ Double-Positive T Cells in the Blood and Liver of Patients during Chronic Hepatitis B and C

PubMed Central

Nascimbeni, Michelina; Pol, Stanislas; Saunier, Bertrand

2011-01-01

CD4+ and CD8+ T cells, the main effectors of adaptive cellular immune responses, differentiate from immature, non-functional CD4+CD8+ double-positive T (DPT) cells in the thymus. Increased proportions of circulating DPT lymphocytes have been observed during acute viral infections; in chronic viral diseases, the role and repartition of extra-thymic DPT cells remain largely uncharacterized. We performed a phenotypic analysis of DPT cells in blood and liver from patients chronically infected by hepatitis C (HCV) or B (HBV) viruses. The highest percentages of DPT cells, predominantly CD4highCD8low, were observed in patients infected by HCV, while HBV-infected patients mostly displayed CD4lowCD8high and CD4highCD8high DPT cells. All proportions of DPT cells were higher in liver than in blood with, for each subpopulation referred to above, a correlation between their frequencies in these two compartments. In HCV patients, intra-hepatic DPT cells displayed more heterogeneous activation, differentiation and memory phenotypes than in the blood; most of them expressed CD1a, a marker of T cell development in the thymus. Ex vivo, the inoculation of liver slices with HCV produced in cell culture was accompanied by a disappearance of CD8high cells, suggesting a direct effect of the virus on the phenotype of DPT cells in the liver. Our results suggest that, in half of the patients, chronic HCV infection promotes the production of DPT cells, perhaps by their re-induction in the thymus and selection in the liver. PMID:21647449

Polyelectrolyte Multilayers in Tissue Engineering

PubMed Central

Detzel, Christopher J.; Larkin, Adam L.

2011-01-01

The layer-by-layer assembly of sequentially adsorbed, alternating polyelectrolytes has become increasingly important over the past two decades. The ease and versatility in assembling polyelectrolyte multilayers (PEMs) has resulted in numerous wide ranging applications of these materials. More recently, PEMs are being used in biological applications ranging from biomaterials, tissue engineering, regenerative medicine, and drug delivery. The ability to manipulate the chemical, physical, surface, and topographical properties of these multilayer architectures by simply changing the pH, ionic strength, thickness, and postassembly modifications render them highly suitable to probe the effects of external stimuli on cellular responsiveness. In the field of regenerative medicine, the ability to sequester growth factors and to tether peptides to PEMs has been exploited to direct the lineage of progenitor cells and to subsequently maintain a desired phenotype. Additional novel applications include the use of PEMs in the assembly of three-dimensional layered architectures and as coatings for individual cells to deliver tunable payloads of drugs or bioactive molecules. This review focuses on literature related to the modulation of chemical and physical properties of PEMs for tissue engineering applications and recent research efforts in maintaining and directing cellular phenotype in stem cell differentiation. PMID:21210759

In vitro mesenchymal trilineage differentiation and extracellular matrix production by adipose and bone marrow derived adult equine multipotent stromal cells on a collagen scaffold.

PubMed

Xie, Lin; Zhang, Nan; Marsano, Anna; Vunjak-Novakovic, Gordana; Zhang, Yanru; Lopez, Mandi J

2013-12-01

Directed differentiation of adult multipotent stromal cells (MSC) is critical for effective treatment strategies. This study was designed to evaluate the capability of equine MSC from bone marrow (BMSC) and adipose tissue (ASC) on a type I collagen (COLI) scaffold to undergo chondrogenic, osteogenic and adipogenic differentiation and form extracellular matrix (ECM) in vitro. Following determination of surface antigen expression, MSC were loaded into scaffolds in a perfusion bioreactor and loading efficiency was quantified. Cell-scaffold constructs were assessed after loading and 7, 14 and 21 days of culture in stromal or induction medium. Cell number was determined with DNA content, cell viability and spatial uniformity with confocal laser microscopy and cell phenotype and matrix production with light and scanning electron microscopy and mRNA levels. The MSC were positive for CD29 (>90 %), CD44 (>99 %), and CD105 (>60 %). Loading efficiencies were >70 %. The ASC and BMSC cell numbers on scaffolds were affected by culture in induction medium differently. Viable cells remained uniformly distributed in scaffolds for up to 21 days and could be directed to differentiate or to maintain an MSC phenotype. Micro- and ultrastructure showed lineage-specific cell and ECM changes. Lineage-specific mRNA levels differed between ASC and BMSC with induction and changed with time. Based on these results, equine ASC and BMSC differentiate into chondrogenic, osteogenic and adipogenic lineages and form ECM similarly on COLI scaffolds. The collected data supports the potential for equine MSC-COLI constructs to support diverse equine tissue formation for controlled biological studies.

Influence of tumors on protective anti-tumor immunity and the effects of irradiation

PubMed Central

Foulds, Gemma A.; Radons, Jürgen; Kreuzer, Mira; Multhoff, Gabriele; Pockley, Alan G.

2012-01-01

Innate and adaptive immunity plays important roles in the development and progression of cancer and it is becoming apparent that tumors can influence the induction of potentially protective responses in a number of ways. The prevalence of immunoregulatory T cell populations in the circulation and tumors of patients with cancer is increased and the presence of these cells appears to present a major barrier to the induction of tumor immunity. One aspect of tumor-mediated immunoregulation which has received comparatively little attention is that which is directed toward natural killer (NK) cells, although evidence that the phenotype and function of NK cell populations are modified in patients with cancer is accumulating. Although the precise mechanisms underlying these localized and systemic immunoregulatory effects remain unclear, tumor-derived factors appear, in part at least, to be involved. The effects could be manifested by an altered function and/or via an influence on the migratory properties of individual cell subsets. A better insight into endogenous immunoregulatory mechanisms and the capacity of tumors to modify the phenotype and function of innate and adaptive immune cells might assist the development of new immunotherapeutic approaches and improve the management of patients with cancer. This article reviews current knowledge relating to the influence of tumors on protective anti-tumor immunity and considers the potential influence that radiation-induced effects might have on the prevalence, phenotype, and function of innate and adaptive immune cells in patients with cancer. PMID:23378947

Integrative Radiation Biology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barcellos-Hoff, Mary Helen

We plan to study tissue-level mechanisms important to human breast radiation carcinogenesis. We propose that the cell biology of irradiated tissues reveals a coordinated multicellular damage response program in which individual cell contributions are primarily directed towards suppression of carcinogenesis and reestablishment of homeostasis. We identified transforming growth factor β1 (TGFβ) as a pivotal signal. Notably, we have discovered that TGFβ suppresses genomic instability by controlling the intrinsic DNA damage response and centrosome integrity. However, TGFβ also mediates disruption of microenvironment interactions, which drive epithelial to mesenchymal transition in irradiated human mammary epithelial cells. This apparent paradox of positive andmore » negative controls by TGFβ is the topic of the present proposal. First, we postulate that these phenotypes manifest differentially following fractionated or chronic exposures; second, that the interactions of multiple cell types in tissues modify the responses evident in this single cell type culture models. The goals are to: 1) study the effect of low dose rate and fractionated radiation exposure in combination with TGFβ on the irradiated phenotype and genomic instability of non-malignant human epithelial cells; and 2) determine whether stromal-epithelial interactions suppress the irradiated phenotype in cell culture and the humanized mammary mouse model. These data will be used to 3) develop a systems biology model that integrates radiation effects across multiple levels of tissue organization and time. Modeling multicellular radiation responses coordinated via extracellular signaling could have a significant impact on the extrapolation of human health risks from high dose to low dose/rate radiation exposure.« less

PDF Receptor Expression Reveals Direct Interactions between Circadian Oscillators in Drosophila

PubMed Central

Im, Seol Hee; Taghert, Paul H.

2010-01-01

Daily rhythms of behavior are controlled by a circuit of circadian pacemaking neurons. In Drosophila, 150 pacemakers participate in this network, and recent observations suggest the network is divisible into M and E oscillators which normally interact and synchronize. Sixteen oscillator neurons (the small and large LNvs) express a neuropeptide called pigment dispersing factor (PDF) whose signaling is often equated with M oscillator output. Given the significance of PDF signaling to numerous aspects of behavioral and molecular rhythms, determining precisely where and how signaling via the PDF receptor (PDFR) occurs is now a central question in the field. Here we show that GAL4-mediated rescue of pdfr phenotypes using a UAS-PDFR transgene is insufficient to provide complete behavioral rescue. In contrast, we describe a ~70 kB PDF receptor (pdfr) transgene which does rescue the entire pdfr circadian behavioral phenotype. The transgene is widely but heterogeneously expressed among pacemakers, and also among a limited number of non-pacemakers. Our results support an important hypothesis: the small LNv cells directly target a subset of the other crucial pacemaker neurons cells. Furthermore, expression of the transgene confirms an autocrine feedback signaling by PDF back to PDF-expressing cells. Finally, the results present an unexpected PDF receptor site: the large LNv cells appear to target a population of non-neuronal cells that resides at the base of the eye. PMID:20394051

Cyclic AMP is a key regulator of M1 to M2a phenotypic conversion of microglia in the presence of Th2 cytokines.

PubMed

Ghosh, Mousumi; Xu, Yong; Pearse, Damien D

2016-01-13

Microglia and macrophages play a central role in neuroinflammation. Pro-inflammatory cytokines trigger their conversion to a classically activated (M1) phenotype, sustaining inflammation and producing a cytotoxic environment. Conversely, anti-inflammatory cytokines polarize the cells towards an alternatively activated (M2), tissue reparative phenotype. Elucidation of the signal transduction pathways involved in M1 to M2 phenotypic conversion may provide insight into how the innate immune response can be harnessed during distinct phases of disease or injury to mediate neuroprotection and neurorepair. Microglial cells (cell line and primary) were subjected to combined cyclic adenosine monophosphate (cyclic AMP) and IL-4, or either alone, in the presence of pro-inflammatory mediators, lipopolysaccharide (LPS), or tumor necrosis factor-α (TNF-α). Their effects on the expression of characteristic markers for M1 and M2 microglia were assessed. Similarly, the M1 and M2 phenotypes of microglia and macrophages within the lesion site were then evaluated following a contusive spinal cord injury (SCI) to the thoracic (T8) spinal cord of rats and mice when the agents were administered systemically. It was demonstrated that cyclic AMP functions synergistically with IL-4 to promote M1 to M2 conversion of microglia in culture. The combination of cyclic AMP and IL-4, but neither alone, induced an Arg-1(+)/iNOS(-)cell phenotype with concomitant expression of other M2-specific markers including TG2 and RELM-α. M2-converted microglia showed ameliorated production of pro-inflammatory cytokines (TNF-α and IP-10) and reactive oxygen species, with no alteration in phagocytic properties. M2a conversion required protein kinase A (PKA), but not the exchange protein directly activated by cyclic AMP (EPAC). Systemic delivery of cyclic AMP and IL-4 after experimental SCI also promoted a significant M1 to M2a phenotypic change in microglia and macrophage population dynamics in the lesion. Using primary microglia, microglial cell lines, and experimental models of CNS injury, we demonstrate that cyclic AMP levels are a critical determinant in M1-M2 polarization. High levels of cyclic AMP promoted an Arg-1(+) M2a phenotype when microglia were activated with pro-inflammatory stimuli and Th2 cytokines. Th2 cytokines or cyclic AMP independently did not promote these changes. Phenotypic conversion of microglia provides a powerful new therapeutic approach for altering the balance of cytotoxic to reparative microglia in a diversity of neurological diseases and injury.

Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation.

PubMed

Marsolier, J; Perichon, M; DeBarry, J D; Villoutreix, B O; Chluba, J; Lopez, T; Garrido, C; Zhou, X Z; Lu, K P; Fritsch, L; Ait-Si-Ali, S; Mhadhbi, M; Medjkane, S; Weitzman, J B

2015-04-16

Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack their genetic and epigenetic machinery to change host cell phenotypic states. Among the Apicomplexa phylum of obligate intracellular parasites, which cause veterinary and human diseases, Theileria is the only genus that transforms its mammalian host cells. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-1 (ref. 2). The transformed phenotypes are reversed by treatment with the theilericidal drug buparvaquone. We used comparative genomics to identify a homologue of the peptidyl-prolyl isomerase PIN1 in T. annulata (TaPIN1) that is secreted into the host cell and modulates oncogenic signalling pathways. Here we show that TaPIN1 is a bona fide prolyl isomerase and that it interacts with the host ubiquitin ligase FBW7, leading to its degradation and subsequent stabilization of c-JUN, which promotes transformation. We performed in vitro and in silico analysis and in vivo zebrafish xenograft experiments to demonstrate that TaPIN1 is directly inhibited by the anti-parasite drug buparvaquone (and other known PIN1 inhibitors) and is mutated in a drug-resistant strain. Prolyl isomerization is thus a conserved mechanism that is important in cancer and is used by Theileria parasites to manipulate host oncogenic signalling.

Mitochondrial “power” drives tamoxifen resistance: NQO1 and GCLC are new therapeutic targets in breast cancer

PubMed Central

Fiorillo, Marco; Sotgia, Federica; Sisci, Diego; Cappello, Anna Rita; Lisanti, Michael P.

2017-01-01

Here, we identified two new molecular targets, which are functionally sufficient to metabolically confer the tamoxifen-resistance phenotype in human breast cancer cells. Briefly, ~20 proteins were first selected as potential candidates, based on unbiased proteomics analysis, using tamoxifen-resistant cell lines. Then, the cDNAs of the most promising candidates were systematically transduced into MCF-7 cells. Remarkably, NQO1 and GCLC were both functionally sufficient to autonomously confer a tamoxifen-resistant metabolic phenotype, characterized by i) increased mitochondrial biogenesis, ii) increased ATP production and iii) reduced glutathione levels. Thus, we speculate that pharmacological inhibition of NQO1 and GCLC may be new therapeutic strategies for overcoming tamoxifen-resistance in breast cancer patients. In direct support of this notion, we demonstrate that treatment with a known NQO1 inhibitor (dicoumarol) is indeed sufficient to revert the tamoxifen-resistance phenotype. As such, these findings could have important translational significance for the prevention of tumor recurrence in ER(+) breast cancers, which is due to an endocrine resistance phenotype. Importantly, we also show here that NQO1 has significant prognostic value as a biomarker for the prediction of tumor recurrence. More specifically, higher levels of NQO1 mRNA strongly predict patient relapse in high-risk ER(+) breast cancer patients receiving endocrine therapy (mostly tamoxifen; H.R. > 2.15; p = 0.007). PMID:28411284

S100A8/A9 regulates MMP-2 expression and invasion and migration by carcinoma cells.

PubMed

Silva, Emmanuel J; Argyris, Prokopios P; Zou, Xianqiong; Ross, Karen F; Herzberg, Mark C

2014-10-01

Intracellular calprotectin (S100A8/A9) functions in the control of the cell cycle checkpoint at G2/M. Dysregulation of S100A8/A9 appears to cause loss of the checkpoint, which frequently characterizes head and neck squamous cell carcinoma (HNSCC). In the present study, we analyzed carcinoma cells for other S100A8/A9-directed changes in malignant phenotype. Using a S100A8/A9-negative human carcinoma cell line (KB), transfection to express S100A8 and S100A9 caused selective down-regulation of MMP-2 and inhibited in vitro invasion and migration. Conversely, silencing of endogenous S100A8 and S100A9 expression in TR146 cells, a well-differentiated HNSCC cell line, increased MMP-2 activity and in vitro invasion and migration. When MMP-2 expression was silenced, cells appeared to assume a less malignant phenotype. To more closely model the architecture of cell growth in vivo, cells were grown in a 3D collagen substrate, which was compared to 2D. Growth on 3D substrates caused greater MMP-2 expression. Whereas hypermethylation of CpG islands occurs frequently in HNSCC, S100A8/A9-dependent regulation of MMP-2 could not be explained by modification of the upstream promoters of MMP2 or TIMP2. Collectively, these results suggest that intracellular S100A8/A9 contributes to the cancer cell phenotype by modulating MMP-2 expression and activity to regulate cell migration and mobility. Published by Elsevier Ltd.

Evolution in health and medicine Sackler colloquium: Stochastic epigenetic variation as a driving force of development, evolutionary adaptation, and disease.

PubMed

Feinberg, Andrew P; Irizarry, Rafael A

2010-01-26

Neo-Darwinian evolutionary theory is based on exquisite selection of phenotypes caused by small genetic variations, which is the basis of quantitative trait contribution to phenotype and disease. Epigenetics is the study of nonsequence-based changes, such as DNA methylation, heritable during cell division. Previous attempts to incorporate epigenetics into evolutionary thinking have focused on Lamarckian inheritance, that is, environmentally directed epigenetic changes. Here, we propose a new non-Lamarckian theory for a role of epigenetics in evolution. We suggest that genetic variants that do not change the mean phenotype could change the variability of phenotype; and this could be mediated epigenetically. This inherited stochastic variation model would provide a mechanism to explain an epigenetic role of developmental biology in selectable phenotypic variation, as well as the largely unexplained heritable genetic variation underlying common complex disease. We provide two experimental results as proof of principle. The first result is direct evidence for stochastic epigenetic variation, identifying highly variably DNA-methylated regions in mouse and human liver and mouse brain, associated with development and morphogenesis. The second is a heritable genetic mechanism for variable methylation, namely the loss or gain of CpG dinucleotides over evolutionary time. Finally, we model genetically inherited stochastic variation in evolution, showing that it provides a powerful mechanism for evolutionary adaptation in changing environments that can be mediated epigenetically. These data suggest that genetically inherited propensity to phenotypic variability, even with no change in the mean phenotype, substantially increases fitness while increasing the disease susceptibility of a population with a changing environment.

Division of labour in the yeast: Saccharomyces cerevisiae.

PubMed

Wloch-Salamon, Dominika M; Fisher, Roberta M; Regenberg, Birgitte

2017-10-01

Division of labour between different specialized cell types is a central part of how we describe complexity in multicellular organisms. However, it is increasingly being recognized that division of labour also plays an important role in the lives of predominantly unicellular organisms. Saccharomyces cerevisiae displays several phenotypes that could be considered a division of labour, including quiescence, apoptosis and biofilm formation, but they have not been explicitly treated as such. We discuss each of these examples, using a definition of division of labour that involves phenotypic variation between cells within a population, cooperation between cells performing different tasks and maximization of the inclusive fitness of all cells involved. We then propose future research directions and possible experimental tests using S. cerevisiae as a model organism for understanding the genetic mechanisms and selective pressures that can lead to the evolution of the very first stages of a division of labour. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Case report and literature review: transient Inab phenotype and an agglutinating anti-IFC in a patient with a gastrointestinal problem.

PubMed

Yazer, Mark H; Judd, W John; Davenport, Robertson D; Dake, Louann R; Lomas-Francis, Christine; Hue-Roye, Kim; Powell, Vivien; Reid, Marion

2006-09-01

The Inab phenotype is a rare deficiency of all Cromer antigens. These antigens are carried on the decay-accelerating factor (DAF, CD55) molecule that is attached to the red blood cell (RBC) membrane by a glycosylphosphatidylinositol (GPI) anchor. Although typically inherited, an acquired and transient form of the Inab phenotype also exists. A patient with the triad of transient Inab phenotype, a direct-agglutinating anti-IFC, and gastrointestinal (GI) abnormalities is reported. An 18-month-old boy with gastroesophageal reflux disease requiring a feeding tube, milk and soy intolerance, and severe growth retardation, as well as vision and hearing deficits from cytomegalovirus infection, was identified when pretransfusion testing revealed a potent panagglutinin (titer > 2000 at 4 degrees C). This antibody did not react with Dr(a-) and IFC RBCs, and the autocontrol was negative. The patient's RBCs lacked CD55 by flow cytometric techniques but had normal levels of CD59 and antigens such as Yt(a) and Emm, carried on GPI-linked proteins, thus excluding paroxysmal nocturnal hemoglobinuria. Several months after initial detection, the anti-IFC was virtually undetectable and his cells reacted weakly with anti-IFC, anti-Dr(a), and anti-CD55. RBCs from the propositus' parents and brother demonstrated normal CD55 and CD59 expression. This is the first example of a direct-agglutinating anti-IFC. The cause of the transient depression in CD55 protein (and thus Cromer system antigens) and appearance of anti-IFC remains unknown, as does the relationship between the patient's GI system abnormalities and these serologic findings.

Xenopatients 2.0

PubMed Central

Menendez, Javier A; Alarcón, Tomás; Corominas-Faja, Bruna; Cuyàs, Elisabet; López-Bonet, Eugeni; Martin, Ángel G; Vellon, Luciano

2014-01-01

In the science-fiction thriller film Minority Report, a specialized police department called “PreCrime” apprehends criminals identified in advance based on foreknowledge provided by 3 genetically altered humans called “PreCogs”. We propose that Yamanaka stem cell technology can be similarly used to (epi)genetically reprogram tumor cells obtained directly from cancer patients and create self-evolving personalized translational platforms to foresee the evolutionary trajectory of individual tumors. This strategy yields a large stem cell population and captures the cancer genome of an affected individual, i.e., the PreCog-induced pluripotent stem (iPS) cancer cells, which are immediately available for experimental manipulation, including pharmacological screening for personalized “stemotoxic” cancer drugs. The PreCog-iPS cancer cells will re-differentiate upon orthotopic injection into the corresponding target tissues of immunodeficient mice (i.e., the PreCrime-iPS mouse avatars), and this in vivo model will run through specific cancer stages to directly explore their biological properties for drug screening, diagnosis, and personalized treatment in individual patients. The PreCog/PreCrime-iPS approach can perform sets of comparisons to directly observe changes in the cancer-iPS cell line vs. a normal iPS cell line derived from the same human genetic background. Genome editing of PreCog-iPS cells could create translational platforms to directly investigate the link between genomic expression changes and cellular malignization that is largely free from genetic and epigenetic noise and provide proof-of-principle evidence for cutting-edge “chromosome therapies” aimed against cancer aneuploidy. We might infer the epigenetic marks that correct the tumorigenic nature of the reprogrammed cancer cell population and normalize the malignant phenotype in vivo. Genetically engineered models of conditionally reprogrammable mice to transiently express the Yamanaka stemness factors following the activation of phenotypic copies of specific cancer diseases might crucially evaluate a “reprogramming cure” for cancer. A new era of xenopatients 2.0 generated via nuclear reprogramming of the epigenetic landscapes of patient-derived cancer genomes might revolutionize the current personalized translational platforms in cancer research. PMID:24406535

Xenopatients 2.0: reprogramming the epigenetic landscapes of patient-derived cancer genomes.

PubMed

Menendez, Javier A; Alarcón, Tomás; Corominas-Faja, Bruna; Cuyàs, Elisabet; López-Bonet, Eugeni; Martin, Angel G; Vellon, Luciano

2014-01-01

In the science-fiction thriller film Minority Report, a specialized police department called "PreCrime" apprehends criminals identified in advance based on foreknowledge provided by 3 genetically altered humans called "PreCogs". We propose that Yamanaka stem cell technology can be similarly used to (epi)genetically reprogram tumor cells obtained directly from cancer patients and create self-evolving personalized translational platforms to foresee the evolutionary trajectory of individual tumors. This strategy yields a large stem cell population and captures the cancer genome of an affected individual, i.e., the PreCog-induced pluripotent stem (iPS) cancer cells, which are immediately available for experimental manipulation, including pharmacological screening for personalized "stemotoxic" cancer drugs. The PreCog-iPS cancer cells will re-differentiate upon orthotopic injection into the corresponding target tissues of immunodeficient mice (i.e., the PreCrime-iPS mouse avatars), and this in vivo model will run through specific cancer stages to directly explore their biological properties for drug screening, diagnosis, and personalized treatment in individual patients. The PreCog/PreCrime-iPS approach can perform sets of comparisons to directly observe changes in the cancer-iPS cell line vs. a normal iPS cell line derived from the same human genetic background. Genome editing of PreCog-iPS cells could create translational platforms to directly investigate the link between genomic expression changes and cellular malignization that is largely free from genetic and epigenetic noise and provide proof-of-principle evidence for cutting-edge "chromosome therapies" aimed against cancer aneuploidy. We might infer the epigenetic marks that correct the tumorigenic nature of the reprogrammed cancer cell population and normalize the malignant phenotype in vivo. Genetically engineered models of conditionally reprogrammable mice to transiently express the Yamanaka stemness factors following the activation of phenotypic copies of specific cancer diseases might crucially evaluate a "reprogramming cure" for cancer. A new era of xenopatients 2.0 generated via nuclear reprogramming of the epigenetic landscapes of patient-derived cancer genomes might revolutionize the current personalized translational platforms in cancer research.

Deterministic Migration-Based Separation of White Blood Cells.

PubMed

Kim, Byeongyeon; Choi, Young Joon; Seo, Hyekyung; Shin, Eui-Cheol; Choi, Sungyoung

2016-10-01

Functional and phenotypic analyses of peripheral white blood cells provide useful clinical information. However, separation of white blood cells from peripheral blood requires a time-consuming, inconvenient process and thus analyses of separated white blood cells are limited in clinical settings. To overcome this limitation, a microfluidic separation platform is developed to enable deterministic migration of white blood cells, directing the cells into designated positions according to a ridge pattern. The platform uses slant ridge structures on the channel top to induce the deterministic migration, which allows efficient and high-throughput separation of white blood cells from unprocessed whole blood. The extent of the deterministic migration under various rheological conditions is explored, enabling highly efficient migration of white blood cells in whole blood and achieving high-throughput separation of the cells (processing 1 mL of whole blood less than 7 min). In the separated cell population, the composition of lymphocyte subpopulations is well preserved, and T cells secrete cytokines without any functional impairment. On the basis of the results, this microfluidic platform is a promising tool for the rapid enrichment of white blood cells, and it is useful for functional and phenotypic analyses of peripheral white blood cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biomaterials patterned with discontinuous microwalls for vascular smooth muscle cell culture: biodegradable small diameter vascular grafts and stable cell culture substrates.

PubMed

Heath, Daniel E; Kang, Gavin C W; Cao, Ye; Poon, Yin Fun; Chan, Vincent; Chan-Park, Mary B

2016-10-01

The medial layer of small diameter blood vessels contains circumferentially aligned vascular smooth muscle cells (vSMC) that possess contractile phenotype. In tissue-engineered constructs, these cellular characteristics are usually achieved by seeding planar scaffolds with vSMC, rolling the cell-laden scaffold into a tubular structure, and maturing the construct in a pulsatile bioreactor, a lengthy process that can take up to two months. During the maturation phase, the cells circumferentially orient, their contractile protein expression increases, and they obtain a contractile phenotype. Generating cell culture platforms that enable the rapid production of directionally oriented vSMC with increased contractile protein expression would be a major step forward for blood vessel tissue engineering and would greatly facilitate the in vitro study of vSMC biology. Previously, we developed a micropatterned cell culture surface that promotes orientation and contractile protein expression of vSMC. Herein, we explore two potential applications of this technology. First, we fabricate tubular and biodegradable scaffolds that possess the micropatterning on their exterior surface. When vSMC are seeded on these scaffolds, they initially proliferate in order to fill the microchannels and as confluence is reached the cells align in the direction of the micropatterning resulting in a biodegradable scaffold that is inhabited by circumferentially aligned vSMC within a week. Second, we illustrate that we can generate biostable cell culture surfaces that allow the in vitro study of the cells in a more contractile state. Specifically, we explore contractile protein expression of cells cultured on the micropatterned surfaces with the addition of soluble transforming growth factor beta one (TGFβ1).

Immunological changes in canine peripheral blood leukocytes triggered by immunization with first or second generation vaccines against canine visceral leishmaniasis.

PubMed

Araújo, Márcio Sobreira Silva; de Andrade, Renata Aline; Sathler-Avelar, Renato; Magalhães, Camila Paula; Carvalho, Andréa Teixeira; Andrade, Mariléia Chaves; Campolina, Sabrina Sidney; Mello, Maria Norma; Vianna, Leonardo Rocha; Mayrink, Wilson; Reis, Alexandre Barbosa; Malaquias, Luiz Cosme Cotta; Rocha, Luciana Morais; Martins-Filho, Olindo Assis

2011-05-15

In this study, we summarized the major phenotypic/functional aspects of circulating leukocytes following canine immunization with Leishvaccine and Leishmune®. Our findings showed that Leishvaccine triggered early changes in the innate immunity (neutrophils and eosinophils) with late alterations on monocytes. Conversely, Leishmune(®) induced early phenotypic changes in both, neutrophils and monocytes. Moreover, Leishvaccine triggered mixed activation-related phenotypic changes on T-cells (CD4+ and CD8+ and B-lymphocytes, whereas Leishmune(®) promoted a selective response, mainly associated with CD8+ T-cell activation. Mixed cytokine profile (IFN-γ/IL-4) was observed in Leishvaccine immunized dogs whereas a selective pro-inflammatory pattern (IFN-γ/NO) was induced by Leishmune® vaccination. The distinct immunological profile triggered by Leishvaccine and Leishmune® may be a direct consequence of the distinct biochemical composition of these immunobiological, i.e. complex versus purified Leishmania antigen along with Bacillus Calmette-Guérin (BCG) versus saponin adjuvant. Both immunobiologicals are able to activate phagocytes and CD8+ T-cells and therefore could be considered as a putative vaccines against canine visceral leishmaniasis (CVL). Copyright © 2011 Elsevier B.V. All rights reserved.

Association of classical markers and establishment of the dyslipidemic sub-phenotype of sickle cell anemia.

PubMed

Aleluia, Milena Magalhães; da Guarda, Caroline Conceição; Santiago, Rayra Pereira; Fonseca, Teresa Cristina Cardoso; Neves, Fábia Idalina; de Souza, Regiana Quinto; Farias, Larissa Alves; Pimenta, Felipe Araújo; Fiuza, Luciana Magalhães; Pitanga, Thassila Nogueira; Ferreira, Júnia Raquel Dutra; Adorno, Elisângela Vitória; Cerqueira, Bruno Antônio Veloso; Gonçalves, Marilda de Souza

2017-04-11

Sickle cell anemia (SCA) patients exhibit sub-phenotypes associated to hemolysis and vaso-occlusion. The disease has a chronic inflammatory nature that has been also associated to alterations in the lipid profile. This study aims to analyze hematological and biochemical parameters to provide knowledge about the SCA sub-phenotypes previously described and suggest a dyslipidemic sub-phenotype. A cross-sectional study was conducted from 2013 to 2014, and 99 SCA patients in steady state were enrolled. We assessed correlations and associations with hematological and biochemical data and investigated the co-inheritance of -α 3.7Kb -thalassemia (-α 3.7Kb -thal). Correlation analyses were performed using Spearman and Pearson coefficient. The median of quantitative variables between two groups was compared using t-test and Mann-Whitney. P-values <0.05 were considered statistically significant. We found significant association of high lactate dehydrogenase levels with decreased red blood cell count and hematocrit as well as high levels of total and indirect bilirubin. SCA patients with low nitric oxide metabolites had high total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol and reduced very low-density cholesterol, triglycerides, direct bilirubin level and reticulocyte counts. In SCA patients with high-density lipoprotein cholesterol greater than 40 mg/dL, we observed increased red blood cell count, hemoglobin, hematocrit, and fetal hemoglobin and decreased nitric oxide metabolites levels. The presence of -α 3.7Kb -thal was associated with high red blood cell count and low mean corpuscular volume, mean corpuscular hemoglobin, platelet count and total and indirect bilirubin levels. Our results provide additional information about the association between biomarkers and co-inheritance of -α 3.7Kb -thal in SCA, and suggest the role of dyslipidemia and nitric oxide metabolites in the characterization of this sub-phenotype.

High-Throughput Phenotyping of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Neurons Using Electric Field Stimulation and High-Speed Fluorescence Imaging

PubMed Central

Daily, Neil J.; Du, Zhong-Wei

2017-01-01

Abstract Electrophysiology of excitable cells, including muscle cells and neurons, has been measured by making direct contact with a single cell using a micropipette electrode. To increase the assay throughput, optical devices such as microscopes and microplate readers have been used to analyze electrophysiology of multiple cells. We have established a high-throughput (HTP) analysis of action potentials (APs) in highly enriched motor neurons and cardiomyocytes (CMs) that are differentiated from human induced pluripotent stem cells (iPSCs). A multichannel electric field stimulation (EFS) device enabled the ability to electrically stimulate cells and measure dynamic changes in APs of excitable cells ultra-rapidly (>100 data points per second) by imaging entire 96-well plates. We found that the activities of both neurons and CMs and their response to EFS and chemicals are readily discerned by our fluorescence imaging-based HTP phenotyping assay. The latest generation of calcium (Ca2+) indicator dyes, FLIPR Calcium 6 and Cal-520, with the HTP device enables physiological analysis of human iPSC-derived samples highlighting its potential application for understanding disease mechanisms and discovering new therapeutic treatments. PMID:28525289

Presence of a non-HLA B cell antigen in rheumatic fever patients and their families as defined by a monoclonal antibody.

PubMed Central

Khanna, A K; Buskirk, D R; Williams, R C; Gibofsky, A; Crow, M K; Menon, A; Fotino, M; Reid, H M; Poon-King, T; Rubinstein, P

1989-01-01

Numerous investigators have suspected that there is a genetic predisposition to rheumatic fever (RF). In this context we have recently produced a series of monoclonal antibodies directed against B cells obtained from RF patients one of which, labeled D8/17, identifies a B cell antigen present in 100% of all RF patients studied. While the highest percentage of positive cells were exhibited by RF probands (33.5% +/- SE), the percentage of cells in unaffected siblings and parents was 14.6 and 13%, respectively. The percentage of positive cells in APSGN probands, unaffected siblings, and parents was 2.96, 3.86, and 2.8%, respectively. A low level of B cells (5-7%) bearing the D8/17 marker was seen in control patients. The segregation pattern of the phenotypes defined by the percentage of D8/17 positive cells within HLA-typed RF families are consistent with an autosomal recessive mode of inheritance not associated with the human MHC system. We postulate that these phenotypes indicate the presence of at least one necessary genetic factor for susceptibility to RF. PMID:2785121

Ex vivo tetramer staining and cell surface phenotyping for early activation markers CD38 and HLA-DR to enumerate and characterize malaria antigen-specific CD8+ T-cells induced in human volunteers immunized with a Plasmodium falciparum adenovirus-vectored malaria vaccine expressing AMA1.

PubMed

Schwenk, Robert; Banania, Glenna; Epstein, Judy; Kim, Yohan; Peters, Bjoern; Belmonte, Maria; Ganeshan, Harini; Huang, Jun; Reyes, Sharina; Stryhn, Anette; Ockenhouse, Christian F; Buus, Soren; Richie, Thomas L; Sedegah, Martha

2013-10-29

Malaria is responsible for up to a 600,000 deaths per year; conveying an urgent need for the development of a malaria vaccine. Studies with whole sporozoite vaccines in mice and non-human primates have shown that sporozoite-induced CD8+ T cells targeting liver stage antigens can mediate sterile protection. There is a need for a direct method to identify and phenotype malaria vaccine-induced CD8+ T cells in humans. Fluorochrome-labelled tetramers consisting of appropriate MHC class I molecules in complex with predicted binding peptides derived from Plasmodium falciparum AMA-1 were used to label ex vivo AMA-1 epitope specific CD8+ T cells from research subjects responding strongly to immunization with the NMRC-M3V-Ad-PfCA (adenovirus-vectored) malaria vaccine. The identification of these CD8+ T cells on the basis of their expression of early activation markers was also investigated. Analyses by flow cytometry demonstrated that two of the six tetramers tested: TLDEMRHFY: HLA-A*01:01 and NEVVVKEEY: HLA-B*18:01, labelled tetramer-specific CD8+ T cells from two HLA-A*01:01 volunteers and one HLA-B*18:01 volunteer, respectively. By contrast, post-immune CD8+ T cells from all six of the immunized volunteers exhibited enhanced expression of the CD38 and HLA-DRhi early activation markers. For the three volunteers with positive tetramer staining, the early activation phenotype positive cells included essentially all of the tetramer positive, malaria epitope- specific CD8+ T cells suggesting that the early activation phenotype could identify all malaria vaccine-induced CD8+ T cells without prior knowledge of their exact epitope specificity. The results demonstrated that class I tetramers can identify ex vivo malaria vaccine antigen-specific CD8+ T cells and could therefore be used to determine their frequency, cell surface phenotype and transcription factor usage. The results also demonstrated that vaccine antigen-specific CD8+ T cells could be identified by activation markers without prior knowledge of their antigen-specificity, using a subunit vaccine for proof-of-concept. Whether, whole parasite or adjuvanted protein vaccines will also induce {CD38 and HLA-DRhi}+ CD8+ T cell populations reflective of the antigen-specific response will the subject of future investigations.

Autophagy dictates metabolism and differentiation of inflammatory immune cells

PubMed Central

Riffelmacher, Thomas; Richter, Felix Clemens; Simon, Anna Katharina

2018-01-01

ABSTRACT The role of macroautophagy/autophagy, a conserved lysosomal degradation pathway, during cellular differentiation has been well studied over the last decade. In particular, evidence for its role during immune cell differentiation is growing. Despite the description of a variety of dramatic immune phenotypes in tissue-specific autophagy knockout models, the underlying mechanisms are still under debate. One of the proposed mechanisms is the impact of autophagy on the altered metabolic states during immune cell differentiation. This concept is strengthened through novel molecular insights into how AMPK and MTOR signaling cascades affect both autophagy and metabolism. In this review, we discuss direct and indirect evidence linking autophagy, metabolic pathways and immune cell differentiation including T, B, and innate lymphocytes as well as in myeloid cells that are direct mediators of inflammation. Herein, we propose a model for autophagy-driven immunometabolism controlling immune cell differentiation. PMID:28806133

HIV-DNA priming alters T-cell responses to HIV-adenovirus vaccine even when responses to DNA are undetectable1

PubMed Central

De Rosa, Stephen C.; Thomas, Evan P.; Bui, John; Huang, Yunda; deCamp, Allan; Morgan, Cecilia; Kalams, Spyros; Tomaras, Georgia D.; Akondy, Rama; Ahmed, Rafi; Lau, Chuen-Yen; Graham, Barney S.; Nabel, Gary J.; McElrath, M. Juliana

2011-01-01

Many candidate HIV vaccines are designed to primarily elicit T-cell responses. Although repeated immunization with the same vaccine boosts antibody responses, the benefit for T-cell responses is ill-defined. We compared two immunization regimens that include the same recombinant adenoviral serotype 5 (rAd5) boost. Repeated homologous rAd5 immunization fails to increase T-cell responses, but increases gp140 antibody responses ten-fold. DNA prime, as compared with rAd5 prime, directs long-term memory CD8+ T cells toward a terminally differentiated effector memory phenotype with cytotoxic potential. Based on the kinetics of activated cells measured directly ex vivo, the DNA vaccination primes for both CD4+ and CD8+ T cells, despite the lack of detection of the latter until after the boost. These results suggest that heterologous prime-boost combinations have distinct immunological advantages over homologous prime-boosts, and suggest that the effect of DNA on subsequent boosting may not be easily detectable directly after the DNA vaccination. PMID:21844392

Improving Therapy of Chronic Lymphocytic Leukemia (CLL) with Chimeric Antigen Receptor (CAR) T Cells

PubMed Central

Fraietta, Joseph A.; Schwab, Robert D.; Maus, Marcela V.

2016-01-01

Adoptive cell immunotherapy for the treatment of chronic lymphocytic leukemia (CLL) has heralded a new era of synthetic biology. The infusion of genetically-engineered, autologous chimeric antigen receptor (CAR) T cells directed against CD19 expressed by normal and malignant B cells represents a novel approach to cancer therapy. The results of recent clinical trials of CAR T cells in relapsed and refractory CLL have demonstrated long-term disease-free remissions, underscoring the power of harnessing and re-directing the immune system against cancer. This review will briefly summarize T cell therapies in development for CLL disease. We discuss the role of T cell function and phenotype, T cell culture optimization, CAR design, and approaches to potentiate the survival and anti-tumor effects of infused lymphocytes. Future efforts will focus on improving the efficacy of CAR T cells for the treatment of CLL and incorporating adoptive cell immunotherapy into standard medical management of CLL. PMID:27040708

A Phenotypic Cell-Binding Screen Identifies a Novel Compound Targeting Triple-Negative Breast Cancer.

PubMed

Chen, Luxi; Long, Chao; Youn, Jonghae; Lee, Jiyong

2018-06-11

We describe a "phenotypic cell-binding screen" by which therapeutic candidate targeting cancer cells of a particular phenotype can be isolated without knowledge of drug targets. Chemical library beads are incubated with cancer cells of the phenotype of interest in the presence of cancer cells lacking the phenotype of interest, and then the beads bound to only cancer cells of the phenotype of interest are selected as hits. We have applied this screening strategy in discovering a novel compound (LC129-8) targeting triple-negative breast cancer (TNBC). LC129-8 displayed highly specific binding to TNBC in cancer cell lines and patient-derived tumor tissues. LC129-8 exerted anti-TNBC activity by inducing apoptosis, inhibiting proliferation, reversing epithelial-mesenchymal transition, downregulating cancer stem cell activity and blocking in vivo tumor growth.

DSE promotes aggressive glioma cell phenotypes by enhancing HB-EGF/ErbB signaling.

PubMed

Liao, Wen-Chieh; Liao, Chih-Kai; Tsai, You-Huan; Tseng, To-Jung; Chuang, Li-Ching; Lan, Chyn-Tair; Chang, Hung-Ming; Liu, Chiung-Hui

2018-01-01

Remodeling of the extracellular matrix (ECM) in the tumor microenvironment promotes glioma progression. Chondroitin sulfate (CS) proteoglycans appear in the ECM and on the cell surface, and can be catalyzed by dermatan sulfate epimerase to form chondroitin sulfate/dermatan sulfate (CS/DS) hybrid chains. Dermatan sulfate epimerase 1 (DSE) is overexpressed in many types of cancer, and CS/DS chains mediate several growth factor signals. However, the role of DSE in gliomas has never been explored. In the present study, we determined the expression of DSE in gliomas by consulting a public database and conducting immunohistochemistry on a tissue array. Our investigation revealed that DSE was upregulated in gliomas compared with normal brain tissue. Furthermore, high DSE expression was associated with advanced tumor grade and poor survival. We found high DSE expression in several glioblastoma cell lines, and DSE expression directly mediated DS chain formation in glioblastoma cells. Knockdown of DSE suppressed the proliferation, migration, and invasion of glioblastoma cells. In contrast, overexpression of DSE in GL261 cells enhanced these malignant phenotypes and in vivo tumor growth. Interestingly, we found that DSE selectively regulated heparin-binding EGF-like growth factor (HB-EGF)-induced signaling in glioblastoma cells. Inhibiting epidermal growth factor receptor (EGFR) and ErbB2 with afatinib suppressed DSE-enhanced malignant phenotypes, establishing the critical role of the ErbB pathway in regulating the effects of DSE expression. This evidence indicates that upregulation of DSE in gliomas contributes to malignant behavior in cancer cells. We provide novel insight into the significance of DS chains in ErbB signaling and glioma pathogenesis.

Live Cell Imaging in Microfluidic Device Proves Resistance to Oxygen/Glucose Deprivation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

PubMed

Martewicz, Sebastian; Gabrel, Giulia; Campesan, Marika; Canton, Marcella; Di Lisa, Fabio; Elvassore, Nicola

2018-05-01

Analyses of cellular responses to fast oxygen dynamics are challenging and require ad hoc technological solutions, especially when decoupling from liquid media composition is required. In this work, we present a microfluidic device specifically designed for culture analyses with high resolution and magnification objectives, providing full optical access to the cell culture chamber. This feature allows fluorescence-based assays, photoactivated surface chemistry, and live cell imaging under tightly controlled pO 2 environments. The device has a simple design, accommodates three independent cell cultures, and can be employed by users with basic cell culture training in studies requiring fast oxygen dynamics, defined media composition, and in-line data acquisition with optical molecular probes. We apply this technology to produce an oxygen/glucose deprived (OGD) environment and analyze cell mortality in murine and human cardiac cultures. Neonatal rat ventricular cardiomyocytes show an OGD time-dependent sensitivity, resulting in a robust and reproducible 66 ± 5% death rate after 3 h of stress. Applying an equivalent stress to human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) provides direct experimental evidence for fetal-like OGD-resistant phenotype. Investigation on the nature of such phenotype exposed large glycogen deposits. We propose a culture strategy aimed at depleting these intracellular energy stores and concurrently activate positive regulation of aerobic metabolic molecular markers. The observed process, however, is not sufficient to induce an OGD-sensitive phenotype in hiPS-CMs, highlighting defective development of mature aerobic metabolism in vitro.

The Biological Role of Nestin(+)-Cells in Physiological and Pathological Cardiovascular Remodeling

PubMed Central

Calderone, Angelino

2018-01-01

The intermediate filament protein nestin was identified in diverse populations of cells implicated in cardiovascular remodeling. Cardiac resident neural progenitor/stem cells constitutively express nestin and following an ischemic insult migrate to the infarct region and participate in angiogenesis and neurogenesis. A modest number of normal adult ventricular fibroblasts express nestin and the intermediate filament protein is upregulated during the progression of reparative and reactive fibrosis. Nestin depletion attenuates cell cycle re-entry suggesting that increased expression of the intermediate filament protein in ventricular fibroblasts may represent an activated phenotype accelerating the biological impact during fibrosis. Nestin immunoreactivity is absent in normal adult rodent ventricular cardiomyocytes. Following ischemic damage, the intermediate filament protein is induced in a modest population of pre-existing adult ventricular cardiomyocytes bordering the peri-infarct/infarct region and nestin(+)-ventricular cardiomyocytes were identified in the infarcted human heart. The appearance of nestin(+)-ventricular cardiomyocytes post-myocardial infarction (MI) recapitulates an embryonic phenotype and depletion of the intermediate filament protein inhibits cell cycle re-entry. Recruitment of the serine/threonine kinase p38 MAPK secondary to an overt inflammatory response after an ischemic insult may represent a seminal event limiting the appearance of nestin(+)-ventricular cardiomyocytes and concomitantly suppressing cell cycle re-entry. Endothelial and vascular smooth muscle cells (VSMCs) express nestin and upregulation of the intermediate filament protein may directly contribute to vascular remodeling. This review will highlight the biological role of nestin(+)-cells during physiological and pathological remodeling of the heart and vasculature and discuss the phenotypic advantage attributed to the intermediate filament protein. PMID:29492403

Loss of tumorigenic potential by human lung tumor cells in the presence of antisense RNA specific to the ectopically synthesized alpha subunit of human chorionic gonadotropin.

PubMed

Rivera, R T; Pasion, S G; Wong, D T; Fei, Y B; Biswas, D K

1989-06-01

A clonal strain of human lung tumor cells in culture (ChaGo), derived from a bronchogenic carcinoma, synthesizes and secretes large amounts of alpha (alpha) and a comparatively lower level of beta (beta) subunit of the glycoprotein hormone, human chorionic gonadotropin (HCG). ChaGo cells lost their characteristic anchorage-independent growth phenotype in the presence of anti-alpha-HCG antibody. The effect of the antibody was partially reversed by addition of alpha-HCG to the culture medium. ChaGo cells were transfected with an expression vector (pRSV-anti-alpha-HCG), that directs synthesis of RNA complementary to alpha-HCG mRNA. The transfectants produced alpha-HCG antisense RNA which was associated with the reduced level of alpha-HCG. Transfectants also displayed several altered phenotypic properties, including altered morphology, less mitosis, reduced growth rate, loss of anchorage-independent growth, and loss of tumorigenicity in nude mice. Treatment of transfectants with 8,bromo-cAMP resulted in increased accumulation of alpha-HCG mRNA, no change in the level of alpha-HCG antisense RNA, release of the inhibition of [3H]thymidine incorporation, and restoration of anchorage-independent growth phenotype. The overexpression of c-myc, observed in ChaGo cells, was unaffected by the reduced level of alpha-HCG. These results suggest that ectopic synthesis of the alpha subunit of HCG plays a functional role in the transformation of these human lung cells.

Cytopathogenesis of vesicular stomatitis virus is regulated by the PSAP motif of M protein in a species-dependent manner.

PubMed

Irie, Takashi; Liu, Yuliang; Drolet, Barbara S; Carnero, Elena; García-Sastre, Adolfo; Harty, Ronald N

2012-09-01

Vesicular stomatitis virus (VSV) is an important vector-borne pathogen of bovine and equine species, causing a reportable vesicular disease. The matrix (M) protein of VSV is multifunctional and plays a key role in cytopathogenesis, apoptosis, host protein shut-off, and virion assembly/budding. Our previous findings indicated that mutations of residues flanking the (37)PSAP(40) motif within the M protein resulted in VSV recombinants having attenuated phenotypes in mice. In this report, we characterize the phenotype of VSV recombinant PS > A4 (which harbors four alanines (AAAA) in place of the PSAP motif without disruption of flanking residues) in both mice, and in Aedes albopictus C6/36 mosquito and Culicoides sonorensis KC cell lines. The PS > A4 recombinant displayed an attenuated phenotype in infected mice as judged by weight loss, mortality, and viral titers measured from lung and brain samples of infected animals. However, unexpectedly, the PS > A4 recombinant displayed a robust cytopathic phenotype in insect C6/36 cells compared to that observed with control viruses. Notably, titers of recombinant PS > A4 were approximately 10-fold greater than those of control viruses in infected C6/36 cells and in KC cells from Culicoides sonorensis, a known VSV vector species. In addition, recombinant PS > A4 induced a 25-fold increase in the level of C3 caspase activity in infected C6/36 cells. These findings indicate that the PSAP motif plays a direct role in regulating cytopathogenicity in a species-dependent manner, and suggest that the intact PSAP motif may be important for maintaining persistence of VSV in an insect host.

Induction of endoplasmic reticulum calcium pump expression during early leukemic B cell differentiation.

PubMed

Aït Ghezali, Lamia; Arbabian, Atousa; Roudot, Hervé; Brouland, Jean-Philippe; Baran-Marszak, Fanny; Salvaris, Evelyn; Boyd, Andrew; Drexler, Hans G; Enyedi, Agnes; Letestu, Remi; Varin-Blank, Nadine; Papp, Bela

2017-06-26

Endoplasmic reticulum (ER) calcium storage and release play important roles in B lymphocyte maturation, survival, antigen-dependent cell activation and immunoglobulin synthesis. Calcium is accumulated in the endoplasmic reticulum (ER) by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes). Because lymphocyte function is critically dependent on SERCA activity, it is important to understand qualitative and quantitative changes of SERCA protein expression that occur during B lymphoid differentiation and leukemogenesis. In this work we investigated the modulation of SERCA expression during the pharmacologically induced differentiation of leukemic precursor B lymphoblast cell lines that carry the E2A-PBX1 fusion oncoprotein. Changes of SERCA levels during differentiation were determined and compared to those of established early B lymphoid differentiation markers. SERCA expression of the cells was compared to that of mature B cell lines as well, and the effect of the direct inhibition of SERCA-dependent calcium transport on the differentiation process was investigated. We show that E2A-PBX1 + leukemia cells simultaneously express SERCA2 and SERCA3-type calcium pumps; however, their SERCA3 expression is markedly inferior to that of mature B cells. Activation of protein kinase C enzymes by phorbol ester leads to phenotypic differentiation of the cells, and this is accompanied by the induction of SERCA3 expression. Direct pharmacological inhibition of SERCA-dependent calcium transport during phorbol ester treatment interferes with the differentiation process. These data show that the calcium pump composition of the ER is concurrent with increased SERCA3 expression during the differentiation of precursor B acute lymphoblastic leukemia cells, that a cross-talk exists between SERCA function and the control of differentiation, and that SERCA3 may constitute an interesting new marker for the study of early B cell phenotype.

Dynamics of phenotypic switching of bacterial cells with temporal fluctuations in pressure

NASA Astrophysics Data System (ADS)

Nepal, Sudip; Kumar, Pradeep

2018-05-01

Phenotypic switching is one of the mechanisms by which bacteria thrive in ever changing environmental conditions around them. Earlier studies have shown that the application of steady high hydrostatic pressure leads to stochastic switching of mesophilic bacteria from a cellular phenotype having a normal cell cycle to another phenotype lacking cell division. Here, we have studied the dynamics of this phenotypic switching with fluctuating periodic pressure using a set of experiments and a theoretical model. Our results suggest that the phenotypic switching rate from high-pressure phenotype to low-pressure phenotype in the reversible regime is larger as compared to the switching rate from low-pressure phenotype to high-pressure phenotype. Furthermore, we find that even though the cell division and elongation are presumably regulated by a large number of genes the underlying physics of the dynamics of stochastic switching at high pressure is captured reasonably well by a simple two-state model.

Interplay between YB-1 and IL-6 promotes the metastatic phenotype in breast cancer cells.

PubMed

Castellana, Bàrbara; Aasen, Trond; Moreno-Bueno, Gema; Dunn, Sandra E; Ramón y Cajal, Santiago

2015-11-10

Epithelial to mesenchymal transition (EMT) induces cell plasticity and promotes metastasis. The multifunctional oncoprotein Y-box binding protein-1 (YB-1) and the pleiotropic cytokine interleukin 6 (IL-6) have both been implicated in tumor cell metastasis and EMT, but via distinct pathways. Here, we show that direct interplay between YB-1 and IL-6 regulates breast cancer metastasis. Overexpression of YB-1 in breast cancer cell lines induced IL-6 production while stimulation with IL-6 increased YB-1 expression and YB-1 phosphorylation. Either approach was sufficient to induce EMT features, including increased cell migration and invasion. Silencing of YB-1 partially reverted the EMT and blocked the effect of IL-6 while inhibition of IL-6 signaling blocked the phenotype induced by YB-1 overexpression, demonstrating a clear YB-1/IL-6 interdependence. Our findings describe a novel signaling network in which YB-1 regulates IL-6, and vice versa, creating a positive feed-forward loop driving EMT-like metastatic features during breast cancer progression. Identification of signaling partners or pathways underlying this co-dependence may uncover novel therapeutic opportunities.

A Method for Identification and Analysis of Non-Overlapping Myeloid Immunophenotypes in Humans

PubMed Central

Gustafson, Michael P.; Lin, Yi; Maas, Mary L.; Van Keulen, Virginia P.; Johnston, Patrick B.; Peikert, Tobias; Gastineau, Dennis A.; Dietz, Allan B.

2015-01-01

The development of flow cytometric biomarkers in human studies and clinical trials has been slowed by inconsistent sample processing, use of cell surface markers, and reporting of immunophenotypes. Additionally, the function(s) of distinct cell types as biomarkers cannot be accurately defined without the proper identification of homogeneous populations. As such, we developed a method for the identification and analysis of human leukocyte populations by the use of eight 10-color flow cytometric protocols in combination with novel software analyses. This method utilizes un-manipulated biological sample preparation that allows for the direct quantitation of leukocytes and non-overlapping immunophenotypes. We specifically designed myeloid protocols that enable us to define distinct phenotypes that include mature monocytes, granulocytes, circulating dendritic cells, immature myeloid cells, and myeloid derived suppressor cells (MDSCs). We also identified CD123 as an additional distinguishing marker for the phenotypic characterization of immature LIN-CD33+HLA-DR- MDSCs. Our approach permits the comprehensive analysis of all peripheral blood leukocytes and yields data that is highly amenable for standardization across inter-laboratory comparisons for human studies. PMID:25799053

Matrix Elasticity of Void-Forming Hydrogels Controls Transplanted Stem Cell-Mediated Bone Formation

PubMed Central

Huebsch, Nathaniel; Lippens, Evi; Lee, Kangwon; Mehta, Manav; Koshy, Sandeep T; Darnell, Max C; Desai, Rajiv; Madl, Christopher M.; Xu, Maria; Zhao, Xuanhe; Chaudhuri, Ovijit; Verbeke, Catia; Kim, Woo Seob; Alim, Karen; Mammoto, Akiko; Ingber, Donald E.; Duda, Georg N; Mooney, David J.

2015-01-01

The effectiveness of stem-cell therapies has been hampered by cell death and limited control over fate1. These problems can be partially circumvented by using macroporous biomaterials that improve the survival of transplanted stem cells and provide molecular cues to direct cell phenotype2–4. Stem cell behavior can also be controlled in vitro by manipulating the elasticity of both porous and non-porous materials5–7, yet translation to therapeutic processes in vivo remains elusive. Here, by developing injectable, void-forming hydrogels that decouple pore formation from elasticity, we show that mesenchymal stem cell (MSC) osteogenesis in vitro, and cell deployment in vitro and in vivo, can be controlled by modifying, respectively, the hydrogel's elastic modulus or its chemistry. When the hydrogels were used to transplant MSCs, the hydrogel's elasticity regulated bone regeneration, with optimal bone formation at 60 kPa. Our findings show that biophysical cues can be harnessed to direct therapeutic stem-cell behaviors in situ. PMID:26366848

Matrix elasticity of void-forming hydrogels controls transplanted-stem-cell-mediated bone formation

NASA Astrophysics Data System (ADS)

Huebsch, Nathaniel; Lippens, Evi; Lee, Kangwon; Mehta, Manav; Koshy, Sandeep T.; Darnell, Max C.; Desai, Rajiv M.; Madl, Christopher M.; Xu, Maria; Zhao, Xuanhe; Chaudhuri, Ovijit; Verbeke, Catia; Kim, Woo Seob; Alim, Karen; Mammoto, Akiko; Ingber, Donald E.; Duda, Georg N.; Mooney, David J.

2015-12-01

The effectiveness of stem cell therapies has been hampered by cell death and limited control over fate. These problems can be partially circumvented by using macroporous biomaterials that improve the survival of transplanted stem cells and provide molecular cues to direct cell phenotype. Stem cell behaviour can also be controlled in vitro by manipulating the elasticity of both porous and non-porous materials, yet translation to therapeutic processes in vivo remains elusive. Here, by developing injectable, void-forming hydrogels that decouple pore formation from elasticity, we show that mesenchymal stem cell (MSC) osteogenesis in vitro, and cell deployment in vitro and in vivo, can be controlled by modifying, respectively, the hydrogel’s elastic modulus or its chemistry. When the hydrogels were used to transplant MSCs, the hydrogel’s elasticity regulated bone regeneration, with optimal bone formation at 60 kPa. Our findings show that biophysical cues can be harnessed to direct therapeutic stem cell behaviours in situ.

Stromagenesis: the changing face of fibroblastic microenvironments during tumor progression.

PubMed

Beacham, Dorothy A; Cukierman, Edna

2005-10-01

During tumorigenesis, reciprocal changes in stromal fibroblasts and tumor cells induce changes to the neoplastic microenvironmental landscape. In stromagenesis, both the complex network of bi-directional stromal fibroblastic signaling pathways and the stromal extracellular matrix are modified. The presence of a 'primed' stroma during the early, reversible stage of tumorigenesis is optimal for stromal-directed therapeutic intervention. Three-dimensional (3D) cell culture systems have been developed that mimic the in vivo microenvironment. These systems provide unique experimental tools to identify early alterations in stromagenesis that are supportive of tumor progression with the ultimate goal of blocking neoplastic permissiveness and restoring normal phenotypes.

A New Wnt1-CRE TomatoRosa Embryonic Stem Cell Line: A Tool for Studying Neural Crest Cell Integration Capacity.

PubMed

Acuna-Mendoza, Soledad; Martin, Sabrina; Kuchler-Bopp, Sabine; Ribes, Sandy; Thalgott, Jérémy; Chaussain, Catherine; Creuzet, Sophie; Lesot, Hervé; Lebrin, Franck; Poliard, Anne

2017-12-01

Neural crest (NC) cells are a migratory, multipotent population giving rise to numerous lineages in the embryo. Their plasticity renders attractive their use in tissue engineering-based therapies, but further knowledge on their in vivo behavior is required before clinical transfer may be envisioned. We here describe the isolation and characterization of a new mouse embryonic stem (ES) line derived from Wnt1-CRE-R26 Rosa TomatoTdv blastocyst and show that it displays the characteristics of typical ES cells. Further, these cells can be efficiently directed toward an NC stem cell-like phenotype as attested by concomitant expression of NC marker genes and Tomato fluorescence. As native NC progenitors, they are capable of differentiating toward typical derivative phenotypes and interacting with embryonic tissues to participate in the formation of neo-structures. Their specific fluorescence allows purification and tracking in vivo. This cellular tool should facilitate a better understanding of the mechanisms driving NC fate specification and help identify the key interactions developed within a tissue after in vivo implantation. Altogether, this novel model may provide important knowledge to optimize NC stem cell graft conditions, which are required for efficient tissue repair.

Interactions between peripheral blood CD8 T lymphocytes and intestinal epithelial cells (iEC)

PubMed Central

Arosa, F A; Irwin, C; Mayer, L; De Sousa, M; Posnett, D N

1998-01-01

Intestinal intraepithelial lymphocytes (iIEL) are primarily CD8 cells and most of them have a CD28− phenotype, the phenotype of effector cytotoxic T cells. We asked whether the predominance of CD8+ CD28− T cells in the gut may result from peripheral blood T cells preferentially migrating to the iIEL compartment and adhering to iEC. Compared with CD4 cells, adhesion of resting CD8+ T cells to iEC cell lines was significantly higher. Adhesion could be blocked with a MoAb to gp180, a molecule expressed on iEC which is known to interact with CD8/lck. No significant difference in the level of adhesion was observed between CD8+ CD28+ and CD8+ CD28− T cells. Thus CD8 cells may preferentially migrate to the iIEL compartment, but loss of CD28 expression could occur in situ after migration. Consistent with this hypothesis, the CD8+ CD28− cells became enriched after co-culturing T cells with iEC cell lines and primary iEC. Induction of the CD8+ CD28− phenotype in cord blood and adult T cells was observed in co-cultures with iEC and also with mitogens and superantigens. In the latter case, CD28 down-modulation was seen specifically in the Vβ subset targeted by the superantigen, indicating that loss of CD28 expression is a direct result of T cell receptor (TCR)-mediated stimulation. The combined results suggest that CD8+ CD28− T cells are antigen experienced T cells, and that they may have a survival advantage in the presence of gut epithelial cells in vitro. This may contribute to the predominance of CD8+ CD28− T cells in the iIEL compartment. PMID:9649184

Anti-inflammatory effects of infliximab in mice are independent of tumour necrosis factor α neutralization.

PubMed

Assas, B M; Levison, S E; Little, M; England, H; Battrick, L; Bagnall, J; McLaughlin, J T; Paszek, P; Else, K J; Pennock, J L

2017-02-01

Infliximab (IFX) has been used repeatedly in mouse preclinical models with associated claims that anti-inflammatory effects are due to inhibition of mouse tumour necrosis factor (TNF)-α. However, the mechanism of action in mice remains unclear. In this study, the binding specificity of IFX for mouse TNF-α was investigated ex vivo using enzyme-linked immunosorbent assay (ELISA), flow cytometry and Western blot. Infliximab (IFX) did not bind directly to soluble or membrane-bound mouse TNF-α nor did it have any effect on TNF-α-induced nuclear factor kappa B (NF-κB) stimulation in mouse fibroblasts. The efficacy of IFX treatment was then investigated in vivo using a TNF-α-independent Trichuris muris-induced infection model of chronic colitis. Infection provoked severe transmural colonic inflammation by day 35 post-infection. Colonic pathology, macrophage phenotype and cell death were determined. As predicted from the in-vitro data, in-vivo treatment of T. muris-infected mice with IFX had no effect on clinical outcome, nor did it affect macrophage cell phenotype or number. IFX enhanced apoptosis of colonic immune cells significantly, likely to be driven by a direct effect of the humanized antibody itself. We have demonstrated that although IFX does not bind directly to TNF-α, observed anti-inflammatory effects in other mouse models may be through host cell apoptosis. We suggest that more careful consideration of xenogeneic responses should be made when utilizing IFX in preclinical models. © 2016 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.

IDH1(R132H) mutation causes a less aggressive phenotype and radiosensitizes human malignant glioma cells independent of the oxygenation status.

PubMed

Kessler, Jacqueline; Güttler, Antje; Wichmann, Henri; Rot, Swetlana; Kappler, Matthias; Bache, Matthias; Vordermark, Dirk

2015-09-01

In malignant glioma the presence of the IDH1 mutation (IDH1(R132H)) is associated with better clinical outcome. However, it is unclear whether IDH1 mutation is associated with a less aggressive phenotype or directly linked to increased sensitivity to radiotherapy. We determined the influence of IDH1(R132H) mutant protein on proliferation and growth in 3D culture, migration, cell survival and radiosensitivity in vitro under normoxia (21% O2) and hypoxia (<1% O2) in a panel of human malignant glioma cell lines (U-251MG, U-343MG, LN-229) with stable overexpression of wild-type (IDH1(wt)) and mutated IDH1 (IDH1(R132H)). Overexpression of IDH1(R132H) in glioma cells resulted in slightly decreased cell proliferation, considerably reduced cell migration and caused differences in growth properties in 3D spheroid cultures. Furthermore, IDH1(R132H)-positive cells consistently demonstrated an increased radiosensitivity in human malignant glioma cells U-251MG (DMF10: 1.52, p<0.01 and 1.42, p<0.01), U-343MG (DMF10: 1.78, p<0.01 and 1.75, p<0.01) and LN-229 (DMF10: 1.41, p<0.05 and 1.68, p<0.01) under normoxia and hypoxia, respectively. Our data indicate that IDH1(R132H) mutation causes both a less aggressive biological behavior and direct radiosensitization of human malignant glioma cells. Targeting IDH1 appears to be an attractive approach in combination with radiotherapy. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

The tumor suppressor TERE1 (UBIAD1) prenyltransferase regulates the elevated cholesterol phenotype in castration resistant prostate cancer by controlling a program of ligand dependent SXR target genes

PubMed Central

Fredericks, William J.; Sepulveda, Jorge; Lal, Priti; Tomaszewski, John E.; Lin, Ming-Fong; McGarvey, Terry; Rauscher, Frank J; Malkowicz, S. Bruce

2013-01-01

Castrate-Resistant Prostate Cancer (CRPC) is characterized by persistent androgen receptor-driven tumor growth in the apparent absence of systemic androgens. Current evidence suggests that CRPC cells can produce their own androgens from endogenous sterol precursors that act in an intracrine manner to stimulate tumor growth. The mechanisms by which CRPC cells become steroidogenic during tumor progression are not well defined. Herein we describe a novel link between the elevated cholesterol phenotype of CRPC and the TERE1 tumor suppressor protein, a prenyltransferase that synthesizes vitamin K-2, which is a potent endogenous ligand for the SXR nuclear hormone receptor. We show that 50% of primary and metastatic prostate cancer specimens exhibit a loss of TERE1 expression and we establish a correlation between TERE1 expression and cholesterol in the LnCaP-C81 steroidogenic cell model of the CRPC. LnCaP-C81 cells also lack TERE1 protein, and show elevated cholesterol synthetic rates, higher steady state levels of cholesterol, and increased expression of enzymes in the de novo cholesterol biosynthetic pathways than the non-steroidogenic prostate cancer cells. C81 cells also show decreased expression of the SXR nuclear hormone receptor and a panel of directly regulated SXR target genes that govern cholesterol efflux and steroid catabolism. Thus, a combination of increased synthesis, along with decreased efflux and catabolism likely underlies the CRPC phenotype: SXR might coordinately regulate this phenotype. Moreover, TERE1 controls synthesis of vitamin K-2, which is a potent endogenous ligand for SXR activation, strongly suggesting a link between TERE1 levels, K-2 synthesis and SXR target gene regulation. We demonstrate that following ectopic TERE1 expression or induction of endogenous TERE1, the elevated cholesterol levels in C81 cells are reduced. Moreover, reconstitution of TERE1 expression in C81 cells reactivates SXR and switches on a suite of SXR target genes that coordinately promote both cholesterol efflux and androgen catabolism. Thus, loss of TERE1 during tumor progression reduces K-2 levels resulting in reduced transcription of SXR target genes. We propose that TERE1 controls the CPRC phenotype by regulating the endogenous levels of Vitamin K-2 and hence the transcriptional control of a suite of steroidogenic genes via the SXR receptor. These data implicate the TERE1 protein as a previously unrecognized link affecting cholesterol and androgen accumulation that could govern acquisition of the CRPC phenotype. PMID:23919967

The use of fluorescence microscopy and image analysis for rapid detection of non-producing revertant cells of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002.

PubMed

Schulze, Katja; Lang, Imke; Enke, Heike; Grohme, Diana; Frohme, Marcus

2015-04-17

Ethanol production via genetically engineered cyanobacteria is a promising solution for the production of biofuels. Through the introduction of a pyruvate decarboxylase and alcohol dehydrogenase direct ethanol production becomes possible within the cells. However, during cultivation genetic instability can lead to mutations and thus loss of ethanol production. Cells then revert back to the wild type phenotype. A method for a rapid and simple detection of these non-producing revertant cells in an ethanol producing cell population is an important quality control measure in order to predict genetic stability and the longevity of a producing culture. Several comparable cultivation experiments revealed a difference in the pigmentation for non-producing and producing cells: the accessory pigment phycocyanin (PC) is reduced in case of the ethanol producer, resulting in a yellowish appearance of the culture. Microarray and western blot studies of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002 confirmed this PC reduction on the level of RNA and protein. Based on these findings we developed a method for fluorescence microscopy in order to distinguish producing and non-producing cells with respect to their pigmentation phenotype. By applying a specific filter set the emitted fluorescence of a producer cell with a reduced PC content appeared orange. The emitted fluorescence of a non-producing cell with a wt pigmentation phenotype was detected in red, and dead cells in green. In an automated process multiple images of each sample were taken and analyzed with a plugin for the image analysis software ImageJ to identify dead (green), non-producing (red) and producing (orange) cells. The results of the presented validation experiments revealed a good identification with 98 % red cells in the wt sample and 90 % orange cells in the producer sample. The detected wt pigmentation phenotype (red cells) in the producer sample were either not fully induced yet (in 48 h induced cultures) or already reverted to a non-producing cells (in long-term photobioreactor cultivations), emphasizing the sensitivity and resolution of the method. The fluorescence microscopy method displays a useful technique for a rapid detection of non-producing single cells in an ethanol producing cell population.

Characterizing human stem cell-derived sensory neurons at the single-cell level reveals their ion channel expression and utility in pain research.

PubMed

Young, Gareth T; Gutteridge, Alex; Fox, Heather DE; Wilbrey, Anna L; Cao, Lishuang; Cho, Lily T; Brown, Adam R; Benn, Caroline L; Kammonen, Laura R; Friedman, Julia H; Bictash, Magda; Whiting, Paul; Bilsland, James G; Stevens, Edward B

2014-08-01

The generation of human sensory neurons by directed differentiation of pluripotent stem cells opens new opportunities for investigating the biology of pain. The inability to generate this cell type has meant that up until now their study has been reliant on the use of rodent models. Here, we use a combination of population and single-cell techniques to perform a detailed molecular, electrophysiological, and pharmacological phenotyping of sensory neurons derived from human embryonic stem cells. We describe the evolution of cell populations over 6 weeks of directed differentiation; a process that results in the generation of a largely homogeneous population of neurons that are both molecularly and functionally comparable to human sensory neurons derived from mature dorsal root ganglia. This work opens the prospect of using pluripotent stem-cell-derived sensory neurons to study human neuronal physiology and as in vitro models for drug discovery in pain and sensory disorders.

Fatty acid metabolic reprogramming via mTOR-mediated inductions of PPARγ directs early activation of T cells

PubMed Central

Angela, Mulki; Endo, Yusuke; Asou, Hikari K.; Yamamoto, Takeshi; Tumes, Damon J.; Tokuyama, Hirotake; Yokote, Koutaro; Nakayama, Toshinori

2016-01-01

To fulfil the bioenergetic requirements for increased cell size and clonal expansion, activated T cells reprogramme their metabolic signatures from energetically quiescent to activated. However, the molecular mechanisms and essential components controlling metabolic reprogramming in T cells are not well understood. Here, we show that the mTORC1–PPARγ pathway is crucial for the fatty acid uptake programme in activated CD4+ T cells. This pathway is required for full activation and rapid proliferation of naive and memory CD4+ T cells. PPARγ directly binds and induces genes associated with fatty acid uptake in CD4+ T cells in both mice and humans. The PPARγ-dependent fatty acid uptake programme is critical for metabolic reprogramming. Thus, we provide important mechanistic insights into the metabolic reprogramming mechanisms that govern the expression of key enzymes, fatty acid metabolism and the acquisition of an activated phenotype during CD4+ T cell activation. PMID:27901044

Hyper-Expression of PD-1 Is Associated with the Levels of Exhausted and Dysfunctional Phenotypes of Circulating CD161++TCR iVα7.2+ Mucosal-Associated Invariant T Cells in Chronic Hepatitis B Virus Infection.

PubMed

Yong, Yean K; Saeidi, Alireza; Tan, Hong Y; Rosmawati, Mohamed; Enström, Philip F; Batran, Rami Al; Vasuki, V; Chattopadhyay, Indranil; Murugesan, Amudhan; Vignesh, Ramachandran; Kamarulzaman, Adeeba; Rajarajeswaran, Jayakumar; Ansari, Abdul W; Vadivelu, Jamuna; Ussher, James E; Velu, Vijayakumar; Larsson, Marie; Shankar, Esaki M

2018-01-01

Mucosal-associated invariant T (MAIT) cells, defined as CD161 ++ TCR iVα7.2 + T cells, play an important role in the innate defense against bacterial infections, and their functionality is impaired in chronic viral infections. Here, we investigated the frequency and functional role of MAIT cells in chronic hepatitis B virus (HBV) infection. The peripheral CD3 + CD161 ++ TCR iVα7.2 + MAIT cells in chronic HBV-infected patients and healthy controls were phenotypically characterized based on CD57, PD-1, TIM-3, and CTLA-4, as well as HLA-DR and CD38 expression. The frequency of MAIT cells was significantly decreased among chronic HBV-infected individuals as compared to controls. Expression of CD57, PD-1, CTLA-4, as well as HLA-DR and CD38 on MAIT cells was significantly elevated in chronic HBV-infected individuals relative to controls. The percentage of T cell receptor (TCR) iVα7.2 + CD161 + MAIT cells did not correlate with HBV viral load but inversely with HLA-DR on CD4 + T cells and MAIT cells and with CD57 on CD8 + T cells suggesting that decrease of MAIT cells may not be attributed to direct infection by HBV but driven by HBV-induced chronic immune activation. The percentage and expression levels of PD-1 as well as CTLA-4 on MAIT cells inversely correlated with plasma HBV-DNA levels, which may suggest either a role for MAIT cells in the control of HBV infection or the effect of HBV replication in the liver on MAIT cell phenotype. We report that decrease of TCR iVα7.2 + MAIT cells in the peripheral blood and their functions were seemingly impaired in chronic HBV-infected patients likely because of the increased expression of PD-1.

MITF and PAX3 Play Distinct Roles in Melanoma Cell Migration; Outline of a "Genetic Switch" Theory Involving MITF and PAX3 in Proliferative and Invasive Phenotypes of Melanoma.

PubMed

Eccles, Michael R; He, Shujie; Ahn, Antonio; Slobbe, Lynn J; Jeffs, Aaron R; Yoon, Han-Seung; Baguley, Bruce C

2013-09-11

Melanoma is a very aggressive neoplasm with a propensity to undergo progression and invasion early in its evolution. The molecular pathways underpinning invasion in melanoma are now just beginning to be elucidated, but a clear understanding of the transition from non-invasive to invasive melanoma cells remains elusive. Microphthalmia-associated transcription factor (MITF), is thought to be a central player in melanoma biology, and it controls many aspects of the phenotypic expression of the melanocytic lineage. However, recently the paired box transcription factor PAX3 was shown to transcriptionally activate POU3F2/BRN2, leading to direct repression of MITF expression. Here we present a theory to explain melanoma phenotype switching and discuss the predictions that this theory makes. One prediction is that independent and opposing roles for MITF and PAX3 in melanoma would be expected, and we present empirical evidence supporting this: in melanoma tissues PAX3 expression occurs independently of MITF, and PAX3 does not play a key role in melanoma cell proliferation. Furthermore, we show that knockdown of PAX3 inhibits cell migration in a group of "lower MITF" melanoma cell lines, while knockdown of MITF promotes cell migration in a complementary "higher MITF" group of melanoma cell lines. Moreover, the morphological effects of knocking down PAX3 versus MITF in melanoma cells were found to differ. While these data support the notion of independent roles for MITF and PAX3, additional experiments are required to provide robust examination of the proposed genetic switch theory. Only upon clear delineation of the mechanisms associated with progression and invasion of melanoma cells will successful treatments for invasive melanoma be developed.

Pre-differentiation of human neural stem cells into GABAergic neurons prior to transplant results in greater repopulation of the damaged brain and accelerates functional recovery after transient ischemic stroke.

PubMed

Abeysinghe, Hima C S; Bokhari, Laita; Quigley, Anita; Choolani, Mahesh; Chan, Jerry; Dusting, Gregory J; Crook, Jeremy M; Kobayashi, Nao R; Roulston, Carli L

2015-09-29

Despite attempts to prevent brain injury during the hyperacute phase of stroke, most sufferers end up with significant neuronal loss and functional deficits. The use of cell-based therapies to recover the injured brain offers new hope. In the current study, we employed human neural stem cells (hNSCs) isolated from subventricular zone (SVZ), and directed their differentiation into GABAergic neurons followed by transplantation to ischemic brain. Pre-differentiated GABAergic neurons, undifferentiated SVZ-hNSCs or media alone were stereotaxically transplanted into the rat brain (n=7/group) 7 days after endothelin-1 induced stroke. Neurological outcome was assessed by neurological deficit scores and the cylinder test. Transplanted cell survival, cellular phenotype and maturation were assessed using immunohistochemistry and confocal microscopy. Behavioral assessments revealed accelerated improvements in motor function 7 days post-transplant in rats treated with pre-differentiated GABAergic cells in comparison to media alone and undifferentiated hNSC treated groups. Histopathology 28 days-post transplant indicated that pre-differentiated cells maintained their GABAergic neuronal phenotype, showed evidence of synaptogenesis and up-regulated expression of both GABA and calcium signaling proteins associated with neurotransmission. Rats treated with pre-differentiated cells also showed increased neurogenic activity within the SVZ at 28 days, suggesting an additional trophic role of these GABAergic cells. In contrast, undifferentiated SVZ-hNSCs predominantly differentiated into GFAP-positive astrocytes and appeared to be incorporated into the glial scar. Our study is the first to show enhanced exogenous repopulation of a neuronal phenotype after stroke using techniques aimed at GABAergic cell induction prior to delivery that resulted in accelerated and improved functional recovery.

Phenotypic Robustness and the Assortativity Signature of Human Transcription Factor Networks

PubMed Central

Pechenick, Dov A.; Payne, Joshua L.; Moore, Jason H.

2014-01-01

Many developmental, physiological, and behavioral processes depend on the precise expression of genes in space and time. Such spatiotemporal gene expression phenotypes arise from the binding of sequence-specific transcription factors (TFs) to DNA, and from the regulation of nearby genes that such binding causes. These nearby genes may themselves encode TFs, giving rise to a transcription factor network (TFN), wherein nodes represent TFs and directed edges denote regulatory interactions between TFs. Computational studies have linked several topological properties of TFNs — such as their degree distribution — with the robustness of a TFN's gene expression phenotype to genetic and environmental perturbation. Another important topological property is assortativity, which measures the tendency of nodes with similar numbers of edges to connect. In directed networks, assortativity comprises four distinct components that collectively form an assortativity signature. We know very little about how a TFN's assortativity signature affects the robustness of its gene expression phenotype to perturbation. While recent theoretical results suggest that increasing one specific component of a TFN's assortativity signature leads to increased phenotypic robustness, the biological context of this finding is currently limited because the assortativity signatures of real-world TFNs have not been characterized. It is therefore unclear whether these earlier theoretical findings are biologically relevant. Moreover, it is not known how the other three components of the assortativity signature contribute to the phenotypic robustness of TFNs. Here, we use publicly available DNaseI-seq data to measure the assortativity signatures of genome-wide TFNs in 41 distinct human cell and tissue types. We find that all TFNs share a common assortativity signature and that this signature confers phenotypic robustness to model TFNs. Lastly, we determine the extent to which each of the four components of the assortativity signature contributes to this robustness. PMID:25121490

PDF receptor expression reveals direct interactions between circadian oscillators in Drosophila.

PubMed

Im, Seol Hee; Taghert, Paul H

2010-06-01

Daily rhythms of behavior are controlled by a circuit of circadian pacemaking neurons. In Drosophila, 150 pacemakers participate in this network, and recent observations suggest that the network is divisible into M and E oscillators, which normally interact and synchronize. Sixteen oscillator neurons (the small and large lateral neurons [LNvs]) express a neuropeptide called pigment-dispersing factor (PDF) whose signaling is often equated with M oscillator output. Given the significance of PDF signaling to numerous aspects of behavioral and molecular rhythms, determining precisely where and how signaling via the PDF receptor (PDFR) occurs is now a central question in the field. Here we show that GAL4-mediated rescue of pdfr phenotypes using a UAS-PDFR transgene is insufficient to provide complete behavioral rescue. In contrast, we describe a approximately 70-kB PDF receptor (pdfr) transgene that does rescue the entire pdfr circadian behavioral phenotype. The transgene is widely but heterogeneously expressed among pacemakers, and also among a limited number of non-pacemakers. Our results support an important hypothesis: the small LNv cells directly target a subset of the other crucial pacemaker neurons cells. Furthermore, expression of the transgene confirms an autocrine feedback signaling by PDF back to PDF-expressing cells. Finally, the results present an unexpected PDF receptor site: the large LNv cells appear to target a population of non-neuronal cells that resides at the base of the eye. (c) 2009 Wiley-Liss, Inc.

Hepatitis C Virus Cell-Cell Transmission and Resistance to Direct-Acting Antiviral Agents

PubMed Central

Heydmann, Laura; Barth, Heidi; Soulier, Eric; Habersetzer, François; Doffoël, Michel; Bukh, Jens; Patel, Arvind H.; Zeisel, Mirjam B.; Baumert, Thomas F.

2014-01-01

Hepatitis C virus (HCV) is transmitted between hepatocytes via classical cell entry but also uses direct cell-cell transfer to infect neighboring hepatocytes. Viral cell-cell transmission has been shown to play an important role in viral persistence allowing evasion from neutralizing antibodies. In contrast, the role of HCV cell-cell transmission for antiviral resistance is unknown. Aiming to address this question we investigated the phenotype of HCV strains exhibiting resistance to direct-acting antivirals (DAAs) in state-of-the-art model systems for cell-cell transmission and spread. Using HCV genotype 2 as a model virus, we show that cell-cell transmission is the main route of viral spread of DAA-resistant HCV. Cell-cell transmission of DAA-resistant viruses results in viral persistence and thus hampers viral eradication. We also show that blocking cell-cell transmission using host-targeting entry inhibitors (HTEIs) was highly effective in inhibiting viral dissemination of resistant genotype 2 viruses. Combining HTEIs with DAAs prevented antiviral resistance and led to rapid elimination of the virus in cell culture model. In conclusion, our work provides evidence that cell-cell transmission plays an important role in dissemination and maintenance of resistant variants in cell culture models. Blocking virus cell-cell transmission prevents emergence of drug resistance in persistent viral infection including resistance to HCV DAAs. PMID:24830295

An in vivo multiplexed small molecule screening platform

PubMed Central

Yang, Dian; Ogasawara, Daisuke; Dix, Melissa M.; Rogers, Zoë N.; Chuang, Chen-Hua; McFarland, Christopher D.; Chiou, Shin-Heng; Brown, J. Mark; Cravatt, Benjamin F.; Bogyo, Matthew; Winslow, Monte M.

2016-01-01

Phenotype-based small molecule screening is a powerful method to identify regulators of cellular function. However, such screens are generally performed in vitro using conditions that do not necessarily model complex physiological conditions or disease states. Here, we use molecular cell barcoding to enable direct in vivo phenotypic screening of libraries of small molecules. The multiplexed nature of this approach allows rapid in vivo analysis of hundreds to thousands of compounds. Using this platform, we screened >700 covalent inhibitors directed towards hydrolases for their effect on pancreatic cancer metastatic seeding. We identified multiple hits and confirmed the relevant target of one compound as the lipase ABHD6. Pharmacological and genetic studies confirmed the role of this enzyme as a regulator of metastatic fitness. Our results highlight the applicability of this multiplexed screening platform for investigating complex processes in vivo. PMID:27617390

Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting: Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4{sup +} T cells compared

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ford, A.L.; Goodsall, A.L.; Sedgwick, J.D.

1995-05-01

Ramified microglia in the adult central nervous system (CNS) are the principal glial element up-regulating MHC class I and II expression in response to inflammatory events or neuronal damage. A proportion of these cells also express MHC class II constitutively in the normal CNS. The role of microglia as APCs for CD4{sup +} cells extravasating into the CNS remains undefined. In this study, using irradiation bone marrow chimeras in CD45-congenic rats, the phenotype CD45{sup low}CD11b/c{sup +} is shown to identify microglial cells specifically within the CNS. Highly purified populations of microglia and nonmicroglial but CNS-associated macrophages (CD45{sup high}CD11b/c{sup +}) havemore » been obtained directly from the adult CNS, by using flow cytometric sorting. Morphologically, freshly isolated microglia vs other CNS macrophages are quite distinct. Of the two populations recovered from the normal CNS, it is the minority CD45{sup high}CD11 b/c{sup +} transitional macrophage population, and not microglia, that is the effective APC for experimental autoimmune encephalomyelitis-inducing CD4{sup +} myelin basic protein (MBP)-reactive T cells. CD45{sup high}CD11b/c{sup +} CNS macrophages also stimulate MBP-reactive T cells without addition of MBP to culture suggesting presentation of endogenous Ag. This is the first study in which microglia vs other CNS macrophages have been analyzed for APC ability directly from the CNS, with substantial cross-contamination between the two populations eliminated. The heterogeneity of these populations in terms of APC function is clearly demonstrated. Evidence is still lacking that adult CNS microglia have the capacity to interact with and stimulate CD4{sup +} T cells to proliferate or secrete IL-2. 60 refs., 6 figs., 1 tab.« less

Phenotypic Plasticity and Cell Fate Decisions in Cancer: Insights from Dynamical Systems Theory.

PubMed

Jia, Dongya; Jolly, Mohit Kumar; Kulkarni, Prakash; Levine, Herbert

2017-06-22

Waddington's epigenetic landscape, a famous metaphor in developmental biology, depicts how a stem cell progresses from an undifferentiated phenotype to a differentiated one. The concept of "landscape" in the context of dynamical systems theory represents a high-dimensional space, in which each cell phenotype is considered as an "attractor" that is determined by interactions between multiple molecular players, and is buffered against environmental fluctuations. In addition, biological noise is thought to play an important role during these cell-fate decisions and in fact controls transitions between different phenotypes. Here, we discuss the phenotypic transitions in cancer from a dynamical systems perspective and invoke the concept of "cancer attractors"-hidden stable states of the underlying regulatory network that are not occupied by normal cells. Phenotypic transitions in cancer occur at varying levels depending on the context. Using epithelial-to-mesenchymal transition (EMT), cancer stem-like properties, metabolic reprogramming and the emergence of therapy resistance as examples, we illustrate how phenotypic plasticity in cancer cells enables them to acquire hybrid phenotypes (such as hybrid epithelial/mesenchymal and hybrid metabolic phenotypes) that tend to be more aggressive and notoriously resilient to therapies such as chemotherapy and androgen-deprivation therapy. Furthermore, we highlight multiple factors that may give rise to phenotypic plasticity in cancer cells, such as (a) multi-stability or oscillatory behaviors governed by underlying regulatory networks involved in cell-fate decisions in cancer cells, and (b) network rewiring due to conformational dynamics of intrinsically disordered proteins (IDPs) that are highly enriched in cancer cells. We conclude by discussing why a therapeutic approach that promotes "recanalization", i.e., the exit from "cancer attractors" and re-entry into "normal attractors", is more likely to succeed rather than a conventional approach that targets individual molecules/pathways.

Induced pluripotent stem cells with a pathological mitochondrial DNA deletion

PubMed Central

Cherry, Anne B. C.; Gagne, Katelyn E.; McLoughlin, Erin M.; Baccei, Anna; Gorman, Bryan; Hartung, Odelya; Miller, Justine D.; Zhang, Jin; Zon, Rebecca L.; Ince, Tan A.; Neufeld, Ellis J.; Lerou, Paul H.; Fleming, Mark D.; Daley, George Q.; Agarwal, Suneet

2013-01-01

In congenital mitochondrial DNA (mtDNA) disorders, a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues, which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown, and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders, as cytoplasmic genetic material is retained during direct reprogramming. Here we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage, we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth, mitochondrial function, and hematopoietic phenotype when differentiated in vitro, compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases. PMID:23400930

High frequencies of Th1-type CD4(+) T cells specific to HTLV-1 Env and Tax proteins in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis.

PubMed

Goon, Peter K C; Hanon, Emmanuel; Igakura, Tadahiko; Tanaka, Yuetsu; Weber, Jonathan N; Taylor, Graham P; Bangham, Charles R M

2002-05-01

CD4(+) T cells are critical for inducing and maintaining efficient humoral and cellular immune responses to pathogens. The CD4(+) T-cell response in human T-lymphotropic virus 1 (HTLV-1) infection has not been studied in detail. However, CD4(+) T cells have been shown to predominate in early lesions in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We present direct estimates of HTLV-1 Env- and Tax-specific CD4(+) T-cell frequencies in patients infected with HTLV-1. We first showed that there was a strong bias toward the Th1 phenotype in these HTLV-1-specific CD4(+) T cells in patients with HAM/TSP. We then demonstrated significantly higher frequencies of HTLV-1-specific Th1-type CD4(+) T cells in HAM/TSP patients than in asymptomatic HTLV-1 carriers. The majority of these HTLV-1-specific CD4(+) T cells did not express HTLV-1 Tax and were therefore unlikely to be infected by HTLV-1. High frequencies of activated HTLV-1-specific CD4(+) T cells of the Th1 phenotype might contribute to the initiation or pathogenesis of HAM/TSP and other HTLV-1-associated inflammatory diseases.

Down-regulation of KIAA1199/CEMIP by miR-216a suppresses tumor invasion and metastasis in colorectal cancer.

PubMed

Zhang, Dejun; Zhao, Lei; Shen, Qiong; Lv, Qing; Jin, Min; Ma, Hong; Nie, Xiu; Zheng, Xiumei; Huang, Shaoyi; Zhou, Pengfei; Wu, Gang; Zhang, Tao

2017-05-15

Colorectal cancer is one of the major causes of death from cancer. Metastasis is the leading cause of treatment failure, in which cancer stem cells and circulating tumor cells play crucial roles. Identifying the involved metastatic biomarkers and clarifying the regulation mechanisms are of great importance for targeting tumor metastasis. In the current research, we discovered that KIAA1199, a cell-migration inducing protein, showed higher expression in CD44+ cancer cells from metastatic compared with the paired primary tissues, and was upregulated in colorectal cancer and positively correlated with numbers and mesenchymal phenotype of circulating tumor cells, and predicted shorter progress-free survival. Moreover, we indicated that down-regulation of KIAA1199 suppressed migration and invasion of colorectal cancer cells in vitro, and inhibited metastasis in vivo. Furthermore, we demonstrated that KIAA1199 was one of the direct and functional targets of miR-216a, and miR-216a overexpression led to decreased migration and invasion of colorectal cancer cells in vitro, and inhibited metastasis in vivo. Collectively, KIAA1199 plays a critical role in maintaining an aggressive phenotype of tumor cells, and suppression of KIAA1199-related motilities of tumor cells contributes to reduced tumor metastasis in colorectal cancer. © 2017 UICC.

A CI-Independent Form of Replicative Inhibition: Turn Off of Early Replication of Bacteriophage Lambda

PubMed Central

Hayes, Sidney; Horbay, Monique A.; Hayes, Connie

2012-01-01

Several earlier studies have described an unusual exclusion phenotype exhibited by cells with plasmids carrying a portion of the replication region of phage lambda. Cells exhibiting this inhibition phenotype (IP) prevent the plating of homo-immune and hybrid hetero-immune lambdoid phages. We have attempted to define aspects of IP, and show that it is directed to repλ phages. IP was observed in cells with plasmids containing a λ DNA fragment including oop, encoding a short OOP micro RNA, and part of the lambda origin of replication, oriλ, defined by iteron sequences ITN1-4 and an adjacent high AT-rich sequence. Transcription of the intact oop sequence from its promoter, pO is required for IP, as are iterons ITN3–4, but not the high AT-rich portion of oriλ. The results suggest that IP silencing is directed to theta mode replication initiation from an infecting repλ genome, or an induced repλ prophage. Phage mutations suppressing IP, i.e., Sip, map within, or adjacent to cro or in O, or both. Our results for plasmid based IP suggest the hypothesis that there is a natural mechanism for silencing early theta-mode replication initiation, i.e. the buildup of λ genomes with oop + oriλ+ sequence. PMID:22590552

dsRNA silencing of an R2R3-MYB transcription factor affects flower cell shape in a Dendrobium hybrid.

PubMed

Lau, Su-Ee; Schwarzacher, Trude; Othman, Rofina Yasmin; Harikrishna, Jennifer Ann

2015-08-11

The R2R3-MYB genes regulate pigmentation and morphogenesis of flowers, including flower and cell shape, and therefore have importance in the development of new varieties of orchids. However, new variety development is limited by the long breeding time required in orchids. In this study, we identified a cDNA, DhMYB1, that is expressed during flower development in a hybrid orchid, Dendrobium hybrida (Dendrobium bobby messina X Dendrobium chao phraya) then used the direct application of dsRNA to observe the effect of gene silencing on flower phenotype and floral epidermal cell shape. Flower bud development in the Dendrobium hybrid was characterised into seven stages and the time of meiosis was determined as between stages 3 to 5 when the bud is approximately half of the mature size. Scanning electron microscopy characterisation of adaxial epidermal cells of the flower perianth, showed that the petals and sepals each are divided into two distinct domains based on cell shape and size, while the labellum comprises seven domains. Thirty-two partial cDNA fragments representing R2R3-MYB gene sequences were isolated from D. hybrida. Phylogenetic analysis revealed that nine of the translated sequences were clustered with MYB sequences that are known to be involved in cell shape development and from these, DhMYB1 was selected for full length cDNA cloning and functional study. Direct application of a 430 bp dsRNA from the 3' region of DhMYB1 to emerging orchid flower buds reduced expression of DhMYB1 RNA compared with untreated control. Scanning electron microscopy of adaxial epidermal cells within domain one of the labellum of flowers treated with DhMYB1 dsRNA showed flattened epidermal cells whilst those of control flowers were conical. DhMYB1 is expressed throughout flower bud development and is involved in the development of the conical cell shape of the epidermal cells of the Dendrobium hybrida flower labellum. The direct application of dsRNA changed the phenotype of floral cells, thus, this technique may have application in floriculture biotechnology.

Identification of immunophenotypic subtypes with different prognoses in extranodal natural killer/T-cell lymphoma, nasal type.

PubMed

Yu, Jian-Bo; Zuo, Zhuo; Zhang, Wen-Yan; Yang, Qun-Pei; Zhang, Ying-Chun; Tang, Yuan; Zhao, Sha; Mo, Xian-Ming; Liu, Wei-Ping

2014-11-01

To analyze the differentiation characteristics of extranodal natural killer/T-cell lymphoma, nasal type, one nude mouse model, cell lines SNK6 and SNT8, and 16 fresh human samples were analyzed by flow cytometry immunophenotyping and immunohistochemistry staining; and 115 archived cases were used for phenotypic detection and prognostic analysis. We found that CD25 was expressed by most tumor cells in all samples, and CD56(+)CD25(+) cells were the predominant population in the mouse model, the 2 cell lines, and 10 of the 16 fresh tumor samples; in the other 6 fresh tumor samples, the predominant cell population was of the CD16(+)CD25(+) phenotype, and only a minor population showed the CD56(+)CD25(+) phenotype. The phenotype detected by immunohistochemistry staining generally was consistent with the phenotype found by flow cytometry immunophenotyping. According to the expression of CD56 and CD16, 115 cases could be classified into 3 phenotypic subtypes: CD56(-)CD16(-), CD56(+)CD16(-), and CD56(dim/-)CD16(+). Patients with tumors of the CD56(dim/-)CD16(+) phenotype had a poorer prognosis than patients with tumors of the other phenotypes. Differentiation of extranodal natural killer/T-cell lymphoma, nasal type apparently resembles the normal natural killer cell developmental pattern, and these tumors can be classified into 3 phenotypic subtypes of different aggressiveness. Expression of CD56(dim/-)CD16(+) implies a poorer prognosis. Copyright © 2014 Elsevier Inc. All rights reserved.

Changing practice: red blood cell typing by molecular methods for patients with sickle cell disease.

PubMed

Casas, Jessica; Friedman, David F; Jackson, Tannoa; Vege, Sunitha; Westhoff, Connie M; Chou, Stella T

2015-06-01

Extended red blood cell (RBC) antigen matching is recommended to limit alloimmunization in patients with sickle cell disease (SCD). DNA-based testing to predict blood group phenotypes has enhanced availability of antigen-negative donor units and improved typing of transfused patients, but replacement of routine serologic typing for non-ABO antigens with molecular typing for patients has not been reported. This study compared the historical RBC antigen phenotypes obtained by hemagglutination methods with genotype predictions in 494 patients with SCD. For discrepant results, repeat serologic testing was performed and/or investigated by gene sequencing for silent or variant alleles. Seventy-one typing discrepancies were identified among 6360 antigen comparisons (1.1%). New specimens for repeat serologic testing were obtained for 66 discrepancies and retyping agreed with the genotype in 64 cases. One repeat Jk(b-) serologic phenotype, predicted Jk(b+) by genotype, was found by direct sequencing of JK to be a silenced allele, and one N typing discrepancy remains under investigation. Fifteen false-negative serologic results were associated with alleles encoding weak antigens or single-dose Fy(b) expression. DNA-based RBC typing provided improved accuracy and expanded information on RBC antigens compared to hemagglutination methods, leading to its implementation as the primary method for extended RBC typing for patients with SCD at our institution. © 2015 AABB.

Variation of Neisseria gonorrhoeae Lipooligosaccharide Directs Dendritic Cell–Induced T Helper Responses

PubMed Central

van Vliet, Sandra J.; Steeghs, Liana; Bruijns, Sven C. M.; Vaezirad, Medi M.; Snijders Blok, Christian; Arenas Busto, Jésus A.; Deken, Marcel; van Putten, Jos P. M.; van Kooyk, Yvette

2009-01-01

Gonorrhea is one of the most prevalent sexually transmitted diseases in the world. A naturally occurring variation of the terminal carbohydrates on the lipooligosaccharide (LOS) molecule correlates with altered disease states. Here, we investigated the interaction of different stable gonoccocal LOS phenotypes with human dendritic cells and demonstrate that each variant targets a different set of receptors on the dendritic cell, including the C-type lectins MGL and DC-SIGN. Neisseria gonorrhoeae LOS phenotype C constitutes the first bacterial ligand to be described for the human C-type lectin receptor MGL. Both MGL and DC-SIGN are locally expressed at the male and female genital area, the primary site of N. gonorrhoeae infection. We show that targeting of different C-type lectins with the N. gonorrhoeae LOS variants results in alterations in dendritic cell cytokine secretion profiles and the induction of distinct adaptive CD4+ T helper responses. Whereas N. gonorrhoeae variant A with a terminal N-acetylglucosamine on its LOS was recognized by DC-SIGN and induced significantly more IL-10 production, phenotype C, carrying a terminal N-acetylgalactosamine, primarily interacted with MGL and skewed immunity towards the T helper 2 lineage. Together, our results indicate that N. gonorrhoeae LOS variation allows for selective manipulation of dendritic cell function, thereby shifting subsequent immune responses in favor of bacterial survival. PMID:19834553

Grass-specific CD4(+) T-cells exhibit varying degrees of cross-reactivity, implications for allergen-specific immunotherapy.

PubMed

Archila, L D; DeLong, J H; Wambre, E; James, E A; Robinson, D M; Kwok, W W

2014-07-01

Conceptually, allergic responses may involve cross-reactivity by antibodies or T-cells. While IgE cross-reactivity among grass-pollen allergens has been observed, cross-reactivity at the allergen-specific T-cell level has been less documented. Identification of the patterns of cross-reactivity may improve our understanding, allowing optimization of better immunotherapy strategies. We use Phleum pratense as model for the studying of cross-reactivity at the allergen-specific CD4(+) T cell level among DR04:01 restricted Pooideae grass-pollen T-cell epitopes. After in vitro culture of blood mono-nucleated cells from grass-pollen-allergic subjects with specific Pooideae antigenic epitopes, dual tetramer staining with APC-labelled DR04:01/Phleum pratense tetramers and PE-labelled DR04:01/Pooideae grass homolog tetramers was assessed to identify cross-reactivity among allergen-specific DR04:01-restricted T-cells in six subjects. Direct ex vivo staining enabled the comparison of frequency and phenotype of different Pooideae grass-pollen reactive T-cells. Intracellular cytokine staining (ICS) assays were also used to examine phenotypes of these T-cells. T-cells with various degrees of cross-reactive profiles could be detected. Poa p 1 97-116 , Lol p 1 221-240 , Lol p 5a 199-218 , and Poa p 5a 199-218 were identified as minimally cross-reactive T-cell epitopes that do not show cross-reactivity to Phl p 1 and Phl p 5a epitopes. Ex vivo tetramer staining assays demonstrated T-cells that recognized these minimally cross-reactive T-cell epitopes are present in Grass-pollen-allergic subjects. Our results suggest that not all Pooideae grass epitopes with sequence homology are cross-reactive. Non-cross-reactive T-cells with comparable frequency, phenotype and functionality to Phl p-specific T-cells suggest that a multiple allergen system should be considered for immunotherapy instead of a mono-allergen system. © 2014 John Wiley & Sons Ltd.

Design and characterization of hybrid peptide sol-gel materials for the solid state induction of neuronal differentiation

NASA Astrophysics Data System (ADS)

Jedlicka, Sabrina S.

2007-12-01

Cell-based therapeutics are a rapidly growing area of research, with considerable promise in the treatment of neurological diseases. One of the primary limitations to neuronal cell-based devices is the necessity to maintain cells in an immature or undifferentiated state in culture prior to transplantation. In many cases, the undifferentiated cell does not express the desired characteristics for implantation. Biologically functional nanomaterials provide the ability to manipulate the direct extracellular environment surrounding cells; influencing their fate and differentiation path. The ability to engineer the interface between the cells and culture materials provides a repeatable, stable means of directing cells down a specific growth path determined by endogenous signaling pathways. This materials approach to cellular engineering can limit the need for added exogenous growth factors, "feeder" layers, or animal sera, in addition to creating a homogenous cell population for transplantation. In this work, hybrid peptide ormosil materials were developed; designed to mimic the developing mammalian brain during corticogenesis. These materials have been developed to enhance the GABAergic phenotype of P19 embryonic carcinoma cells and immature immortalized neurons. The ability to develop a homogenous, directed cell population has implications in stem cell research, regenerative medicine, cell-based devices and biosensing technology.

The MAZ transcription factor is a downstream target of the oncoprotein Cyr61/CCN1 and promotes pancreatic cancer cell invasion via CRAF-ERK signaling.

PubMed

Maity, Gargi; Haque, Inamul; Ghosh, Arnab; Dhar, Gopal; Gupta, Vijayalaxmi; Sarkar, Sandipto; Azeem, Imaan; McGregor, Douglas; Choudhary, Abhishek; Campbell, Donald R; Kambhampati, Suman; Banerjee, Sushanta K; Banerjee, Snigdha

2018-03-23

Myc-associated zinc-finger protein (MAZ) is a transcription factor with dual roles in transcription initiation and termination. Deregulation of MAZ expression is associated with the progression of pancreatic ductal adenocarcinoma (PDAC). However, the mechanism of action of MAZ in PDAC progression is largely unknown. Here, we present evidence that MAZ mRNA expression and protein levels are increased in human PDAC cell lines, tissue samples, a subcutaneous tumor xenograft in a nude mouse model, and spontaneous cancer in the genetically engineered PDAC mouse model. We also found that MAZ is predominantly expressed in pancreatic cancer stem cells. Functional analysis indicated that MAZ depletion in PDAC cells inhibits invasive phenotypes such as the epithelial-to-mesenchymal transition, migration, invasion, and the sphere-forming ability of PDAC cells. Mechanistically, we detected no direct effects of MAZ on the expression of K-Ras mutants, but MAZ increased the activity of CRAF-ERK signaling, a downstream signaling target of K-Ras. The MAZ-induced activation of CRAF-ERK signaling was mediated via p21-activated protein kinase (PAK) and protein kinase B (AKT/PKB) signaling cascades and promoted PDAC cell invasiveness. Moreover, we found that the matricellular oncoprotein cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) regulates MAZ expression via Notch-1-sonic hedgehog signaling in PDAC cells. We propose that Cyr61/CCN1-induced expression of MAZ promotes invasive phenotypes of PDAC cells not through direct K-Ras activation but instead through the activation of CRAF-ERK signaling. Collectively, these results highlight key molecular players in PDAC invasiveness and may help inform therapeutic strategies to improve clinical management and outcomes of PDAC.

Fast Raman single bacteria identification: toward a routine in-vitro diagnostic

NASA Astrophysics Data System (ADS)

Douet, Alice; Josso, Quentin; Marchant, Adrien; Dutertre, Bertrand; Filiputti, Delphine; Novelli-Rousseau, Armelle; Espagnon, Isabelle; Kloster-Landsberg, Meike; Mallard, Frédéric; Perraut, Francois

2016-04-01

Timely microbiological results are essential to allow clinicians to optimize the prescribed treatment, ideally at the initial stage of the therapeutic process. Several approaches have been proposed to solve this issue and to provide the microbiological result in a few hours directly from the sample such as molecular biology. However fast and sensitive those methods are not based on single phenotypic information which presents several drawbacks and limitations. Optical methods have the advantage to allow single-cell sensitivity and to probe the phenotype of measured cells. Here we present a process and a prototype that allow automated single-bacteria phenotypic analysis. This prototype is based on the use of Digital In-line Holography techniques combined with a specially designed Raman spectrometer using a dedicated device to capture bacteria. The localization of single-cell is finely determined by using holograms and a proper propagation kernel. Holographic images are also used to analyze bacteria in the sample to sort potential pathogens from flora dwelling species or other biological particles. This accurate localization enables the use of a small confocal volume adapted to the measurement of single-cell. Along with the confocal volume adaptation, we also have modified every components of the spectrometer to optimize single-bacteria Raman measurements. This optimization allowed us to acquire informative single-cell spectra using an integration time of 0.5s only. Identification results obtained with this prototype are presented based on a 65144 Raman spectra database acquired automatically on 48 bacteria strains belonging to 8 species.

Unsupervised automated high throughput phenotyping of RNAi time-lapse movies.

PubMed

Failmezger, Henrik; Fröhlich, Holger; Tresch, Achim

2013-10-04

Gene perturbation experiments in combination with fluorescence time-lapse cell imaging are a powerful tool in reverse genetics. High content applications require tools for the automated processing of the large amounts of data. These tools include in general several image processing steps, the extraction of morphological descriptors, and the grouping of cells into phenotype classes according to their descriptors. This phenotyping can be applied in a supervised or an unsupervised manner. Unsupervised methods are suitable for the discovery of formerly unknown phenotypes, which are expected to occur in high-throughput RNAi time-lapse screens. We developed an unsupervised phenotyping approach based on Hidden Markov Models (HMMs) with multivariate Gaussian emissions for the detection of knockdown-specific phenotypes in RNAi time-lapse movies. The automated detection of abnormal cell morphologies allows us to assign a phenotypic fingerprint to each gene knockdown. By applying our method to the Mitocheck database, we show that a phenotypic fingerprint is indicative of a gene's function. Our fully unsupervised HMM-based phenotyping is able to automatically identify cell morphologies that are specific for a certain knockdown. Beyond the identification of genes whose knockdown affects cell morphology, phenotypic fingerprints can be used to find modules of functionally related genes.

Tissue-engineered vascular grafts composed of marine collagen and PLGA fibers using pulsatile perfusion bioreactors.

PubMed

Jeong, Sung In; Kim, So Yeon; Cho, Seong Kwan; Chong, Moo Sang; Kim, Kyung Soo; Kim, Hyuck; Lee, Sang Bong; Lee, Young Moo

2007-02-01

Novel tubular scaffolds of marine source collagen and PLGA fibers were fabricated by freeze drying and electrospinning processes for vascular grafts. The hybrid scaffolds, composed of a porous collagen matrix and a fibrous PLGA layer, had an average pore size of 150+/-50 microm. The electrospun fibrous PLGA layer on the surface of a porous tubular collagen scaffold improved the mechanical strength of the collagen scaffolds in both the dry and wet states. Smooth muscle cells (SMCs)- and endothelial cells (ECs)-cultured collagen/PLGA scaffolds exhibited mechanical properties similar to collagen/PLGA scaffolds unseeded with cells, even after culturing for 23 days. The effect of a mechanical stimulation on the proliferation and phenotype of SMCs and ECs, cultured on collagen/PLGA scaffolds, was evaluated. The pulsatile perfusion system enhanced the SMCs and ECs proliferation. In addition, a significant cell alignment in a direction radial to the distending direction was observed in tissues exposed to radial distention, which is similar to the phenomenon of native vessel tissues in vivo. On the other hand, cells in tissues engineered in the static condition were randomly aligned. Immunochemical analyses showed that the expressions of SM alpha-actin, SM myosin heavy chain, EC von Willebrand factor, and EC nitric oxide were upregulated in tissues engineered under a mechano-active condition, compared to vessel tissues engineered in the static condition. These results indicated that the co-culturing of SMCs and ECs, using collagen/PLGA hybrid scaffolds under a pulsatile perfusion system, leads to the enhancement of vascular EC development, as well as the retention of the differentiated cell phenotype.

β-Cell Ca(2+) dynamics and function are compromised in aging.

PubMed

Barker, Christopher J; Li, Luosheng; Köhler, Martin; Berggren, Per-Olof

2015-01-01

Defects in pancreatic β-cell function and survival are key components in type 2 diabetes (T2D). An age-dependent deterioration in β-cell function has also been observed, but little is known about the molecular mechanisms behind this phenomenon. Our previous studies indicate that the regulation of cytoplasmic free Ca(2+) concentration ([Ca(2+)]i) may be critical and that this is dependent on the proper function of the mitochondria. The [Ca(2+)]i dynamics of the pancreatic β-cell are driven by an interplay between glucose-induced influx of extracellular Ca(2+) via voltage-dependent Ca(2+) channels and the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-mediated liberation of Ca(2+) from intracellular stores. Our previous work has indicated a direct relationship between disruption of Ins(1,4,5)P3-mediated Ca(2+) regulation and loss of β-cell function, including disturbed [Ca(2+)]i dynamics and compromised insulin secretion. To investigate these processes in aging we used three mouse models, a premature aging mitochondrial mutator mouse, a mature aging phenotype (C57BL/6) and an aging-resistant phenotype (129). Our data suggest that age-dependent impairment in mitochondrial function leads to modest changes in [Ca(2+)]i dynamics in mouse β-cells, particularly in the pattern of [Ca(2+)]i oscillations. These changes are driven by modifications in both PLC/Ins(1,4,5)P3-mediated Ca(2+) mobilization from intracellular stores and decreased β-cell Ca(2+) influx over the plasma membrane. Our findings underscore an important concept, namely that even relatively small, time-dependent changes in β-cell signal-transduction result in compromised insulin release and in a diabetic phenotype. Copyright © 2014 Elsevier Ltd. All rights reserved.

Differentiation of human adipose-derived stem cells co-cultured with urothelium cell line toward a urothelium-like phenotype in a nude murine model.

PubMed

Zhang, Ming; Peng, Yubing; Zhou, Zhe; Zhou, Juan; Wang, Zhong; Lu, Mujun

2013-02-01

To investigated the urothelium differentiation potential of adipose-derived stem cells (ASCs) that were coimplanted with the immortalized human bladder urothelium cell line (SV-HUC-1) into the subcutaneous tissue of athymic mice. The ASCs were isolated from the human adipose tissue of patients undergoing liposuction procedures and were expanded in vitro. After labeling with CM-DiI, the ASCs were mixed with SV-HUC-1 and implanted into the subcutaneous tissue of athymic mice for 2 and 4 weeks. The urothelium-specific markers uroplakin-Ia and uroplakin-II were detected by immunofluorescence. The transformation rate of ASCs into the urothelium phenotype was evaluated at each measurement point. We found that 25.87% ± 1.38% of ASCs expressed the urothelium-specific marker uroplakin-Ia and 23.60% ± 2.57% of ASCs expressed uroplakin-II 2 weeks after coimplantation with SV-HUC-1 in vivo. After 4 weeks, 70.07% ± 3.84% of ASCs expressed uroplakin-Ia and 65.56% ± 2.94% expressed uroplakin-II. However, no obvious organizational multilayered urothelium structure, such as that of the native bladder mucosa, was found in the subcutaneous tissues of the athymic mice. The results of our study have demonstrated that ASCs could be differentiated toward the urothelium-like phenotype when they were coimplanted in direct contact with cells of a mature urothelium cell line, and the proportion of differentiated cells increased from 2 to 4 weeks. The differentiation potential of ASCs toward the urothelial cell type suggests that ASCs might have potential to be used in urinary tract repair with a tissue engineering approach in the future. Copyright © 2013 Elsevier Inc. All rights reserved.

Poliovirus Mutants Resistant to Neutralization with Soluble Cell Receptors

NASA Astrophysics Data System (ADS)

Kaplan, Gerardo; Peters, David; Racaniello, Vincent R.

1990-12-01

Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.

Macrophage phenotypic subtypes diametrically regulate epithelial-mesenchymal plasticity in breast cancer cells.

PubMed

Yang, Min; Ma, Bo; Shao, Hanshuang; Clark, Amanda M; Wells, Alan

2016-07-07

Metastatic progression of breast cancer involves phenotypic plasticity of the carcinoma cells moving between epithelial and mesenchymal behaviors. During metastatic seeding and dormancy, even highly aggressive carcinoma cells take on an E-cadherin-positive epithelial phenotype that is absent from the emergent, lethal metastatic outgrowths. These phenotypes are linked to the metastatic microenvironment, though the specific cells and induction signals are still to be deciphered. Recent evidence suggests that macrophages impact tumor progression, and may alter the balance between cancer cell EMT and MErT in the metastatic microenvironment. Here we explore the role of M1/M2 macrophages in epithelial-mesenchymal plasticity of breast cancer cells by coculturing epithelial and mesenchymal cells lines with macrophages. We found that after polarizing the THP-1 human monocyte cell line, the M1 and M2-types were stable and maintained when co-cultured with breast cancer cells. Surprisingly, M2 macrophages may conferred a growth advantage to the epithelial MCF-7 cells, with these cells being driven to a partial mesenchymal phenotypic as indicated by spindle morphology. Notably, E-cadherin protein expression is significantly decreased in MCF-7 cells co-cultured with M2 macrophages. M0 and M1 macrophages had no effect on the MCF-7 epithelial phenotype. However, the M1 macrophages impacted the highly aggressive mesenchymal-like MDA-MB-231 breast cancer cells to take on a quiescent, epithelial phenotype with re-expression of E-cadherin. The M2 macrophages if anything exacerbated the mesenchymal phenotype of the MDA-MB-231 cells. Our findings demonstrate M2 macrophages might impart outgrowth and M1 macrophages may contribute to dormancy behaviors in metastatic breast cancer cells. Thus EMT and MErT are regulated by selected macrophage phenotype in the liver metastatic microenvironment. These results indicate macrophage could be a potential therapeutic target for limiting death due to malignant metastases in breast cancer.

Single-cell mechanical phenotype is an intrinsic marker of reprogramming and differentiation along the mouse neural lineage.

PubMed

Urbanska, Marta; Winzi, Maria; Neumann, Katrin; Abuhattum, Shada; Rosendahl, Philipp; Müller, Paul; Taubenberger, Anna; Anastassiadis, Konstantinos; Guck, Jochen

2017-12-01

Cellular reprogramming is a dedifferentiation process during which cells continuously undergo phenotypical remodeling. Although the genetic and biochemical details of this remodeling are fairly well understood, little is known about the change in cell mechanical properties during the process. In this study, we investigated changes in the mechanical phenotype of murine fetal neural progenitor cells (fNPCs) during reprogramming to induced pluripotent stem cells (iPSCs). We find that fNPCs become progressively stiffer en route to pluripotency, and that this stiffening is mirrored by iPSCs becoming more compliant during differentiation towards the neural lineage. Furthermore, we show that the mechanical phenotype of iPSCs is comparable with that of embryonic stem cells. These results suggest that mechanical properties of cells are inherent to their developmental stage. They also reveal that pluripotent cells can differentiate towards a more compliant phenotype, which challenges the view that pluripotent stem cells are less stiff than any cells more advanced developmentally. Finally, our study indicates that the cell mechanical phenotype might be utilized as an inherent biophysical marker of pluripotent stem cells. © 2017. Published by The Company of Biologists Ltd.

Epithelial Cell Invasion and Adherence Directed by the Enterotoxigenic Escherichia coli tib Locus Is Associated with a 104-Kilodalton Outer Membrane Protein

DTIC Science & Technology

1994-08-01

probes. The organisms examined in this required for the adherence and invasion phenotypes of the tib study included Aeromonas hydrophila, Bordetella ... pertussis , locus. The position of the ibA gene within the tib locus was Campylobacter jejuni, Citrobacter freundii, Klebsiella pneu- identified by T7

Coregulation of srGAP1 by Wnt and Androgen Receptor Signaling: A New Target for Treatment of CRPC

DTIC Science & Technology

2015-10-01

Specific Aim 1: Test the... Specific Aim2: Test the hypothesis that down regulating srGAP1 in CRPC cells change phenotypic...direct interaction between AR and β-catenin seemed to elicit a specific expression of a set of target genes in low androgen conditions in CRPC.

Target identification of small molecules based on chemical biology approaches.

PubMed

Futamura, Yushi; Muroi, Makoto; Osada, Hiroyuki

2013-05-01

Recently, a phenotypic approach-screens that assess the effects of compounds on cells, tissues, or whole organisms-has been reconsidered and reintroduced as a complementary strategy of a target-based approach for drug discovery. Although the finding of novel bioactive compounds from large chemical libraries has become routine, the identification of their molecular targets is still a time-consuming and difficult process, making this step rate-limiting in drug development. In the last decade, we and other researchers have amassed a large amount of phenotypic data through progress in omics research and advances in instrumentation. Accordingly, the profiling methodologies using these datasets expertly have emerged to identify and validate specific molecular targets of drug candidates, attaining some progress in current drug discovery (e.g., eribulin). In the case of a compound that shows an unprecedented phenotype likely by inhibiting a first-in-class target, however, such phenotypic profiling is invalid. Under the circumstances, a photo-crosslinking affinity approach should be beneficial. In this review, we describe and summarize recent progress in both affinity-based (direct) and phenotypic profiling (indirect) approaches for chemical biology target identification.

Highly efficient gene inactivation by adenoviral CRISPR/Cas9 in human primary cells

PubMed Central

Tielen, Frans; Elstak, Edo; Benschop, Julian; Grimbergen, Max; Stallen, Jan; Janssen, Richard; van Marle, Andre; Essrich, Christian

2017-01-01

Phenotypic assays using human primary cells are highly valuable tools for target discovery and validation in drug discovery. Expression knockdown (KD) of such targets in these assays allows the investigation of their role in models of disease processes. Therefore, efficient and fast modes of protein KD in phenotypic assays are required. The CRISPR/Cas9 system has been shown to be a versatile and efficient means of gene inactivation in immortalized cell lines. Here we describe the use of adenoviral (AdV) CRISPR/Cas9 vectors for efficient gene inactivation in two human primary cell types, normal human lung fibroblasts and human bronchial epithelial cells. The effects of gene inactivation were studied in the TGF-β-induced fibroblast to myofibroblast transition assay (FMT) and the epithelial to mesenchymal transition assay (EMT), which are SMAD3 dependent and reflect pathogenic mechanisms observed in fibrosis. Co-transduction (co-TD) of AdV Cas9 with SMAD3-targeting guide RNAs (gRNAs) resulted in fast and efficient genome editing judged by insertion/deletion (indel) formation, as well as significant reduction of SMAD3 protein expression and nuclear translocation. This led to phenotypic changes downstream of SMAD3 inhibition, including substantially decreased alpha smooth muscle actin and fibronectin 1 expression, which are markers for FMT and EMT, respectively. A direct comparison between co-TD of separate Cas9 and gRNA AdV, versus TD with a single “all-in-one” Cas9/gRNA AdV, revealed that both methods achieve similar levels of indel formation. These data demonstrate that AdV CRISPR/Cas9 is a useful and efficient tool for protein KD in human primary cell phenotypic assays. The use of AdV CRISPR/Cas9 may offer significant advantages over the current existing tools and should enhance target discovery and validation opportunities. PMID:28800587

In-depth evaluation of commercially available human vascular smooth muscle cells phenotype: Implications for vascular tissue engineering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Timraz, Sara B.H., E-mail: sara.timraz@kustar.ac.ae; Farhat, Ilyas A.H., E-mail: ilyas.farhat@outlook.com; Alhussein, Ghada, E-mail: ghada.alhussein@kustar.ac.ae

In vitro research on vascular tissue engineering has extensively used isolated primary human or animal smooth muscle cells (SMC). Research programs that lack such facilities tend towards commercially available primary cells sources. Here, we aim to evaluate the capacity of commercially available human SMC to maintain their contractile phenotype, and determine if dedifferentiation towards the synthetic phenotype occurs in response to conventional cell culture and passaging without any external biochemical or mechanical stimuli. Lower passage SMC adopted a contractile phenotype marked by a relatively slower proliferation rate, higher expression of proteins of the contractile apparatus and smoothelin, elongated morphology, andmore » reduced deposition of collagen types I and III. As the passage number increased, migratory capacity was enhanced, average cell speed, total distance and net distance travelled increased up to passage 8. Through the various assays, corroborative evidence pinpoints SMC at passage 7 as the transition point between the contractile and synthetic phenotypes, while passage 8 distinctly and consistently exhibited characteristics of synthetic phenotype. This knowledge is particularly useful in selecting SMC of appropriate passage number for the target vascular tissue engineering application, for example, a homeostatic vascular graft for blood vessel replacement versus recreating atherosclerotic blood vessel model in vitro. - Highlights: • Ability of human smooth muscle cells to alter phenotype in culture is evaluated. • Examined the effect of passaging human smooth muscle cells on phenotype. • Phenotype is assessed based on morphology, proliferation, markers, and migration. • Multi-resolution assessment methodology, single-cell and cell-population. • Lower and higher passages than P7 adopted a contractile and synthetic phenotype respectively.« less

A novel spiroindoline targets cell cycle and migration via modulation of microtubule cytoskeleton.

PubMed

Kumar, Naveen; Hati, Santanu; Munshi, Parthapratim; Sen, Subhabrata; Sehrawat, Seema; Singh, Shailja

2017-05-01

Natural product-inspired libraries of molecules with diverse architectures have evolved as one of the most useful tools for discovering lead molecules for drug discovery. In comparison to conventional combinatorial libraries, these molecules have been inferred to perform better in phenotypic screening against complicated targets. Diversity-oriented synthesis (DOS) is a forward directional strategy to access such multifaceted library of molecules. From a successful DOS campaign of a natural product-inspired library, recently a small molecule with spiroindoline motif was identified as a potent anti-breast cancer compound. Herein we report the subcellular studies performed for this molecule on breast cancer cells. Our investigation revealed that it repositions microtubule cytoskeleton and displaces AKAP9 located at the microtubule organization centre. DNA ladder assay and cell cycle experiments further established the molecule as an apoptotic agent. This work further substantiated the amalgamation of DOS-phenotypic screening-sub-cellular studies as a consolidated blueprint for the discovery of potential pharmaceutical drug candidates.

PPARs: Interference with Warburg' Effect and Clinical Anticancer Trials

PubMed Central

Vamecq, Joseph; Colet, Jean-Marie; Vanden Eynde, Jean Jacques; Briand, Gilbert; Porchet, Nicole; Rocchi, Stéphane

2012-01-01

The metabolic/cell signaling basis of Warburg's effect (“aerobic glycolysis”) and the general metabolic phenotype adopted by cancer cells are first reviewed. Several bypasses are adopted to provide a panoramic integrated view of tumoral metabolism, by attributing a central signaling role to hypoxia-induced factor (HIF-1) in the expression of aerobic glycolysis. The cancer metabolic phenotype also results from alterations of other routes involving ras, myc, p53, and Akt signaling and the propensity of cancer cells to develop signaling aberrances (notably aberrant surface receptor expression) which, when present, offer unique opportunities for therapeutic interventions. The rationale for various emerging strategies for cancer treatment is presented along with mechanisms by which PPAR ligands might interfere directly with tumoral metabolism and promote anticancer activity. Clinical trials using PPAR ligands are reviewed and followed by concluding remarks and perspectives for future studies. A therapeutic need to associate PPAR ligands with other anticancer agents is perhaps an important lesson to be learned from the results of the clinical trials conducted to date. PMID:22654896

Studying Cancer Stem Cell Dynamics on PDMS Surfaces for Microfluidics Device Design

PubMed Central

Zhang, Weijia; Choi, Dong Soon; Nguyen, Yen H.; Chang, Jenny; Qin, Lidong

2013-01-01

This systematic study clarified a few interfacial aspects of cancer cell phenotypes on polydimethylsiloxane (PDMS) substrates and indicated that the cell phenotypic equilibrium greatly responds to cell-to-surface interactions. We demonstrated that coatings of fibronectin, bovine serum albumin (BSA), or collagen with or without oxygen-plasma treatments of the PDMS surfaces dramatically impacted the phenotypic equilibrium of breast cancer stem cells, while the variations of the PDMS elastic stiffness had much less such effects. Our results showed that the surface coatings of collagen and fibronectin on PDMS maintained breast cancer cell phenotypes to be nearly identical to the cultures on commercial polystyrene Petri dishes. The surface coating of BSA provided a weak cell-substrate adhesion that stimulated the increase in stem-cell-like subpopulation. Our observations may potentially guide surface modification approaches to obtain specific cell phenotypes. PMID:23900274

Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

PubMed

Alvarado-Vazquez, Perla Abigail; Bernal, Laura; Paige, Candler A; Grosick, Rachel L; Moracho Vilrriales, Carolina; Ferreira, David Wilson; Ulecia-Morón, Cristina; Romero-Sandoval, E Alfonso

2017-08-01

M1 macrophages release proinflammatory factors during inflammation. They transit to an M2 phenotype and release anti-inflammatory factors to resolve inflammation. An imbalance in the transition from M1 to M2 phenotype in macrophages contributes to the development of persistent inflammation. CD163, a member of the scavenger receptor cysteine-rich family, is an M2 macrophage marker. The functional role of CD163 during the resolution of inflammation is not completely known. We postulate that CD163 contributes to the transition from M1 to M2 phenotype in macrophages. We induced CD163 gene in THP-1 and primary human macrophages using polyethylenimine nanoparticles grafted with a mannose ligand (Man-PEI). This nanoparticle specifically targets cells of monocytic origin via mannose receptors. Cells were challenged with a single or a double stimulation of lipopolysaccharide (LPS). A CD163 or empty plasmid was complexed with Man-PEI nanoparticles for cell transfections. Quantitative RT-PCR, immunocytochemistry, and ELISAs were used for molecular assessments. CD163-overexpressing macrophages displayed reduced levels of tumor necrosis factor-alpha (TNF)-α and monocytes chemoattractant protein (MCP)-1 after a single stimulation with LPS. Following a double stimulation paradigm, CD163-overexpressing macrophages showed an increase of interleukin (IL)-10 and IL-1ra and a reduction of MCP-1. This anti-inflammatory phenotype was partially blocked by an anti-CD163 antibody (effects on IL-10 and IL-1ra). A decrease in the release of TNF-α, IL-1β, and IL-6 was observed in CD163-overexpressing human primary macrophages. The release of IL-6 was blocked by an anti-CD163 antibody in the CD163-overexpressing group. Our data show that the induction of the CD163 gene in human macrophages under inflammatory conditions produces changes in cytokine secretion in favor of an anti-inflammatory phenotype. Targeting macrophages to induce CD163 using cell-directed nanotechnology is an attractive and practical approach for inflammatory conditions that could lead to persistent pain, i.e. major surgeries, burns, rheumatoid arthritis, etc. Copyright © 2017 Elsevier GmbH. All rights reserved.

Innate Immune Cytokines, Fibroblast Phenotypes, and Regulation of Extracellular Matrix in Lung.

PubMed

Richards, Carl D

2017-02-01

Chronic inflammation can be caused by adaptive immune responses in autoimmune and allergic conditions, driven by a T lymphocyte subset balance (TH1, TH2, Th17, Th22, and/or Treg) and skewed cellular profiles in an antigen-specific manner. However, several chronic inflammatory diseases have no clearly defined adaptive immune mechanisms that drive chronicity. These conditions include those that affect the lung such as nonatopic asthma or idiopathic pulmonary fibrosis comprising significant health problems. The remodeling of extracellular matrix (ECM) causes organ dysfunction, and it is largely generated by fibroblasts as the major cell controlling net ECM. As such, these are potential targets of treatment approaches in the context of ECM pathology. Fibroblast phenotypes contribute to ECM and inflammatory cell accumulation, and they are integrated into chronic disease mechanisms including cancer. Evidence suggests that innate cytokine responses may be critical in nonallergic/nonautoimmune disease, and they enable environmental agent exposure mechanisms that are independent of adaptive immunity. Innate immune cytokines derived from macrophage subsets (M1/M2) and innate lymphoid cell (ILC) subsets can directly regulate fibroblast function. We also suggest that STAT3-activating gp130 cytokines can sensitize fibroblasts to the innate cytokine milieu to drive phenotypes and exacerbate existing adaptive responses. Here, we review evidence exploring innate cytokine regulation of fibroblast behavior.

Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation

PubMed Central

Marsolier, J.; Perichon, M.; DeBarry, JD.; Villoutreix, BO.; Chluba, J.; Lopez, T.; Garrido, C.; Zhou, XZ.; Lu, KP.; Fritsch, L.; Ait-Si-Ali, S.; Mhadhbi, M; Medjkane, S.; Weitzman, JB.

2014-01-01

Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack the genetic and epigenetic machinery to change phenotypic states. Amongst the Apicomplexa phylum of obligate intracellular parasites which cause veterinary and human diseases, Theileria is the only genus which transforms its mammalian host cells1. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-12. The transformed phenotypes are reversed by treatment with the theilericidal drug Buparvaquone3. We used comparative genomics to identify a homologue of the Peptidyl Prolyl Isomerase Pin1 (designated TaPin1) in T. annulata which is secreted into the host cell and modulates oncogenic signalling pathways. Here we show that TaPin1 is a bona fide prolyl isomerase and that it interacts with the host ubiquitin ligase FBW7 leading to its degradation and subsequent stabilization of c-Jun which promotes transformation. We performed in vitro analysis and in vivo zebrafish xenograft experiments to demonstrate that TaPin1 is directly inhibited by the anti-parasite drug Buparvaquone (and other known Pin1 inhibitors) and is mutated in a drug-resistant strain. Prolyl isomerisation is thus a conserved mechanism which is important in cancer and is used by Theileria parasites to manipulate host oncogenic signaling. PMID:25624101

Downregulation of GbpB, a Component of the VicRK Regulon, Affects Biofilm Formation and Cell Surface Characteristics of Streptococcus mutans▿ †

PubMed Central

Duque, Cristiane; Stipp, Rafael N.; Wang, Bing; Smith, Daniel J.; Höfling, José F.; Kuramitsu, Howard K.; Duncan, Margaret J.; Mattos-Graner, Renata O.

2011-01-01

The virulence of the dental caries pathogen Streptococcus mutans relies in part on the sucrose-dependent synthesis of and interaction with glucan, a major component of the extracellular matrix of tooth biofilms. However, the mechanisms by which secreted and/or cell-associated glucan-binding proteins (Gbps) produced by S. mutans participate in biofilm growth remain to be elucidated. In this study, we further investigate GbpB, an essential immunodominant protein with similarity to murein hydrolases. A conditional knockdown mutant that expressed gbpB antisense RNA under the control of a tetracycline-inducible promoter was constructed in strain UA159 (UACA2) and used to investigate the effects of GbpB depletion on biofilm formation and cell surface-associated characteristics. Additionally, regulation of gbpB by the two-component system VicRK was investigated, and phenotypic analysis of a vicK mutant (UAvicK) was performed. GbpB was directly regulated by VicR, and several phenotypic changes were comparable between UACA2 and UAvicK, although differences between these strains existed. It was established that GbpB depletion impaired initial phases of sucrose-dependent biofilm formation, while exogenous native GbpB partially restored the biofilm phenotype. Several cellular traits were significantly affected by GbpB depletion, including altered cell shape, decreased autolysis, increased cell hydrophobicity, and sensitivity to antibiotics and osmotic and oxidative stresses. These data provide the first experimental evidence for GbpB participation in sucrose-dependent biofilm formation and in cell surface properties. PMID:21078847

Morphological and functional aspects of progenitors perturbed in cortical malformations

PubMed Central

Bizzotto, Sara; Francis, Fiona

2015-01-01

In this review, we discuss molecular and cellular mechanisms important for the function of neuronal progenitors during development, revealed by their perturbation in different cortical malformations. We focus on a class of neuronal progenitors, radial glial cells (RGCs), which are renowned for their unique morphological and behavioral characteristics, constituting a key element during the development of the mammalian cerebral cortex. We describe how the particular morphology of these cells is related to their roles in the orchestration of cortical development and their influence on other progenitor types and post-mitotic neurons. Important for disease mechanisms, we overview what is currently known about RGC cellular components, cytoskeletal mechanisms, signaling pathways and cell cycle characteristics, focusing on how defects lead to abnormal development and cortical malformation phenotypes. The multiple recent entry points from human genetics and animal models are contributing to our understanding of this important cell type. Combining data from phenotypes in the mouse reveals molecules which potentially act in common pathways. Going beyond this, we discuss future directions that may provide new data in this expanding area. PMID:25729350

Automatic analysis of dividing cells in live cell movies to detect mitotic delays and correlate phenotypes in time.

PubMed

Harder, Nathalie; Mora-Bermúdez, Felipe; Godinez, William J; Wünsche, Annelie; Eils, Roland; Ellenberg, Jan; Rohr, Karl

2009-11-01

Live-cell imaging allows detailed dynamic cellular phenotyping for cell biology and, in combination with small molecule or drug libraries, for high-content screening. Fully automated analysis of live cell movies has been hampered by the lack of computational approaches that allow tracking and recognition of individual cell fates over time in a precise manner. Here, we present a fully automated approach to analyze time-lapse movies of dividing cells. Our method dynamically categorizes cells into seven phases of the cell cycle and five aberrant morphological phenotypes over time. It reliably tracks cells and their progeny and can thus measure the length of mitotic phases and detect cause and effect if mitosis goes awry. We applied our computational scheme to annotate mitotic phenotypes induced by RNAi gene knockdown of CKAP5 (also known as ch-TOG) or by treatment with the drug nocodazole. Our approach can be readily applied to comparable assays aiming at uncovering the dynamic cause of cell division phenotypes.

Glucose-induced gradual phenotypic modulation of cultured human glomerular epithelial cells may be independent of Wilms’ tumor 1 (WT1)

PubMed Central

2013-01-01

Background Renal podocytes form the main filtration barrier possessing a unique phenotype maintained by proteins including podocalyxin and nephrin, the expression of which is suppressed in pathological conditions. We used an in vitro model of human glomerular epithelial cells (HGEC) to investigate the role of high glucose in dysregulating the podocytic epithelial phenotype and determined the time needed for this change to occur. Results In our in vitro podocyte system changes indicating podocyte dedifferentiation in the prolonged presence of high glucose included loss of podocalyxin, nephrin and CD10/CALLA concomitant with upregulation of mesenchymal vimentin. Our study demonstrates for the first time that podocyte-specific markers undergo changes of expression at different time intervals, since glucose-mediated podocalyxin downregulation is a progressive process that precedes downregulation of nephrin expression. Finally we demonstrate that high glucose permanently impaired WT1 binding to the podocalyxin gene promoter region but did not affect WT1 binding on the nephrin gene promoter region. Conclusion The presence of high glucose induced a phenotypic conversion of podocytes resembling partial dedifferentiation. Our study demonstrates that dysregulation of the normal podocytic phenotype is an event differentially affecting the expression of function-specific podocytic markers, exhibiting downregulation of the epithelial marker CD10/CALLA and PC first, followed by stably downregulated nephrin. Furthermore, it is herein suggested that WT1 may not be directly involved with upregulation of previously reduced PC and nephrin expression. PMID:23768159

Iterative sorting reveals CD133+ and CD133- melanoma cells as phenotypically distinct populations.

PubMed

Grasso, Carole; Anaka, Matthew; Hofmann, Oliver; Sompallae, Ramakrishna; Broadley, Kate; Hide, Winston; Berridge, Michael V; Cebon, Jonathan; Behren, Andreas; McConnell, Melanie J

2016-09-09

The heterogeneity and tumourigenicity of metastatic melanoma is attributed to a cancer stem cell model, with CD133 considered to be a cancer stem cell marker in melanoma as well as other tumours, but its role has remained controversial. We iteratively sorted CD133+ and CD133- cells from 3 metastatic melanoma cell lines, and observed tumourigenicity and phenotypic characteristics over 7 generations of serial xeno-transplantation in NOD/SCID mice. We demonstrate that iterative sorting is required to make highly pure populations of CD133+ and CD133- cells from metastatic melanoma, and that these two populations have distinct characteristics not related to the cancer stem cell phenotype. In vitro, gene set enrichment analysis indicated CD133+ cells were related to a proliferative phenotype, whereas CD133- cells were of an invasive phenotype. However, in vivo, serial transplantation of CD133+ and CD133- tumours over 7 generations showed that both populations were equally able to initiate and propagate tumours. Despite this, both populations remained phenotypically distinct, with CD133- cells only able to express CD133 in vivo and not in vitro. Loss of CD133 from the surface of a CD133+ cell was observed in vitro and in vivo, however CD133- cells derived from CD133+ retained the CD133+ phenotype, even in the presence of signals from the tumour microenvironment. We show for the first time the necessity of iterative sorting to isolate pure marker-positive and marker-negative populations for comparative studies, and present evidence that despite CD133+ and CD133- cells being equally tumourigenic, they display distinct phenotypic differences, suggesting CD133 may define a distinct lineage in melanoma.

Inferring fitness landscapes and selection on phenotypic states from single-cell genealogical data

PubMed Central

Kussell, Edo

2017-01-01

Recent advances in single-cell time-lapse microscopy have revealed non-genetic heterogeneity and temporal fluctuations of cellular phenotypes. While different phenotypic traits such as abundance of growth-related proteins in single cells may have differential effects on the reproductive success of cells, rigorous experimental quantification of this process has remained elusive due to the complexity of single cell physiology within the context of a proliferating population. We introduce and apply a practical empirical method to quantify the fitness landscapes of arbitrary phenotypic traits, using genealogical data in the form of population lineage trees which can include phenotypic data of various kinds. Our inference methodology for fitness landscapes determines how reproductivity is correlated to cellular phenotypes, and provides a natural generalization of bulk growth rate measures for single-cell histories. Using this technique, we quantify the strength of selection acting on different cellular phenotypic traits within populations, which allows us to determine whether a change in population growth is caused by individual cells’ response, selection within a population, or by a mixture of these two processes. By applying these methods to single-cell time-lapse data of growing bacterial populations that express a resistance-conferring protein under antibiotic stress, we show how the distributions, fitness landscapes, and selection strength of single-cell phenotypes are affected by the drug. Our work provides a unified and practical framework for quantitative measurements of fitness landscapes and selection strength for any statistical quantities definable on lineages, and thus elucidates the adaptive significance of phenotypic states in time series data. The method is applicable in diverse fields, from single cell biology to stem cell differentiation and viral evolution. PMID:28267748

Nuclear PTEN functions as an essential regulator of SRF-dependent transcription to control smooth muscle differentiation

PubMed Central

Horita, Henrick; Wysoczynski, Christina L.; Walker, Lori A.; Moulton, Karen S.; Li, Marcella; Ostriker, Allison; Tucker, Rebecca; McKinsey, Timothy A.; Churchill, Mair E. A.; Nemenoff, Raphael A.; Weiser-Evans, Mary C. M.

2016-01-01

Vascular disease progression is associated with marked changes in vascular smooth muscle cell (SMC) phenotype and function. SMC contractile gene expression and, thus differentiation, is under direct transcriptional control by the transcription factor, serum response factor (SRF); however, the mechanisms dynamically regulating SMC phenotype are not fully defined. Here we report that the lipid and protein phosphatase, PTEN, has a novel role in the nucleus by functioning as an indispensible regulator with SRF to maintain the differentiated SM phenotype. PTEN interacts with the N-terminal domain of SRF and PTEN–SRF interaction promotes SRF binding to essential promoter elements in SM-specific genes. Factors inducing phenotypic switching promote loss of nuclear PTEN through nucleo-cytoplasmic translocation resulting in reduced myogenically active SRF, but enhanced SRF activity on target genes involved in proliferation. Overall decreased expression of PTEN was observed in intimal SMCs of human atherosclerotic lesions underlying the potential clinical importance of these findings. PMID:26940659

Nuclear PTEN functions as an essential regulator of SRF-dependent transcription to control smooth muscle differentiation.

PubMed

Horita, Henrick; Wysoczynski, Christina L; Walker, Lori A; Moulton, Karen S; Li, Marcella; Ostriker, Allison; Tucker, Rebecca; McKinsey, Timothy A; Churchill, Mair E A; Nemenoff, Raphael A; Weiser-Evans, Mary C M

2016-03-04

Vascular disease progression is associated with marked changes in vascular smooth muscle cell (SMC) phenotype and function. SMC contractile gene expression and, thus differentiation, is under direct transcriptional control by the transcription factor, serum response factor (SRF); however, the mechanisms dynamically regulating SMC phenotype are not fully defined. Here we report that the lipid and protein phosphatase, PTEN, has a novel role in the nucleus by functioning as an indispensible regulator with SRF to maintain the differentiated SM phenotype. PTEN interacts with the N-terminal domain of SRF and PTEN-SRF interaction promotes SRF binding to essential promoter elements in SM-specific genes. Factors inducing phenotypic switching promote loss of nuclear PTEN through nucleo-cytoplasmic translocation resulting in reduced myogenically active SRF, but enhanced SRF activity on target genes involved in proliferation. Overall decreased expression of PTEN was observed in intimal SMCs of human atherosclerotic lesions underlying the potential clinical importance of these findings.

Discovery of a “White-Gray-Opaque” Tristable Phenotypic Switching System in Candida albicans: Roles of Non-genetic Diversity in Host Adaptation

PubMed Central

Guan, Guobo; Dai, Yu; Nobile, Clarissa J.; Liang, Weihong; Cao, Chengjun; Zhang, Qiuyu; Zhong, Jin; Huang, Guanghua

2014-01-01

Non-genetic phenotypic variations play a critical role in the adaption to environmental changes in microbial organisms. Candida albicans, a major human fungal pathogen, can switch between several morphological phenotypes. This ability is critical for its commensal lifestyle and for its ability to cause infections. Here, we report the discovery of a novel morphological form in C. albicans, referred to as the “gray” phenotype, which forms a tristable phenotypic switching system with the previously reported white and opaque phenotypes. White, gray, and opaque cell types differ in a number of aspects including cellular and colony appearances, mating competency, secreted aspartyl proteinase (Sap) activities, and virulence. Of the three cell types, gray cells exhibit the highest Sap activity and the highest ability to cause cutaneous infections. The three phenotypes form a tristable phenotypic switching system, which is independent of the regulation of the mating type locus (MTL). Gray cells mate over 1,000 times more efficiently than do white cells, but less efficiently than do opaque cells. We further demonstrate that the master regulator of white-opaque switching, Wor1, is essential for opaque cell formation, but is not required for white-gray transitions. The Efg1 regulator is required for maintenance of the white phenotype, but is not required for gray-opaque transitions. Interestingly, the wor1/wor1 efg1/efg1 double mutant is locked in the gray phenotype, suggesting that Wor1 and Efg1 could function coordinately and play a central role in the regulation of gray cell formation. Global transcriptional analysis indicates that white, gray, and opaque cells exhibit distinct gene expression profiles, which partly explain their differences in causing infections, adaptation ability to diverse host niches, metabolic profiles, and stress responses. Therefore, the white-gray-opaque tristable phenotypic switching system in C. albicans may play a significant role in a wide range of biological aspects in this common commensal and pathogenic fungus. PMID:24691005

Discovery of a "white-gray-opaque" tristable phenotypic switching system in candida albicans: roles of non-genetic diversity in host adaptation.

PubMed

Tao, Li; Du, Han; Guan, Guobo; Dai, Yu; Nobile, Clarissa J; Liang, Weihong; Cao, Chengjun; Zhang, Qiuyu; Zhong, Jin; Huang, Guanghua

2014-04-01

Non-genetic phenotypic variations play a critical role in the adaption to environmental changes in microbial organisms. Candida albicans, a major human fungal pathogen, can switch between several morphological phenotypes. This ability is critical for its commensal lifestyle and for its ability to cause infections. Here, we report the discovery of a novel morphological form in C. albicans, referred to as the "gray" phenotype, which forms a tristable phenotypic switching system with the previously reported white and opaque phenotypes. White, gray, and opaque cell types differ in a number of aspects including cellular and colony appearances, mating competency, secreted aspartyl proteinase (Sap) activities, and virulence. Of the three cell types, gray cells exhibit the highest Sap activity and the highest ability to cause cutaneous infections. The three phenotypes form a tristable phenotypic switching system, which is independent of the regulation of the mating type locus (MTL). Gray cells mate over 1,000 times more efficiently than do white cells, but less efficiently than do opaque cells. We further demonstrate that the master regulator of white-opaque switching, Wor1, is essential for opaque cell formation, but is not required for white-gray transitions. The Efg1 regulator is required for maintenance of the white phenotype, but is not required for gray-opaque transitions. Interestingly, the wor1/wor1 efg1/efg1 double mutant is locked in the gray phenotype, suggesting that Wor1 and Efg1 could function coordinately and play a central role in the regulation of gray cell formation. Global transcriptional analysis indicates that white, gray, and opaque cells exhibit distinct gene expression profiles, which partly explain their differences in causing infections, adaptation ability to diverse host niches, metabolic profiles, and stress responses. Therefore, the white-gray-opaque tristable phenotypic switching system in C. albicans may play a significant role in a wide range of biological aspects in this common commensal and pathogenic fungus.

Antitumor activity and inhibitory effects on cancer stem cell-like properties of Adeno-associated virus (AAV) -mediated Bmi-1 interference driven by Bmi-1 promoter for gastric cancer

PubMed Central

Wang, Xiaofeng; Liu, Xinyang; Huang, Mingzhu; Gan, Lu; Cheng, Yufan; Li, Jin

2016-01-01

Bmi-1 is aberrantly activated in various cancers and plays a vital role in maintaining the self-renewal of stem cells. Our previous research revealed that Bmi-1 was overexpressed in gastric cancer (GC) and it's overexpression was an independent negative prognostic factor, suggesting it can be a therapeutic target. The main purpose of this investigation was to explore the antitumor activity of Bmi-1 interference driven by its own promoter (Ad-Bmi-1i) for GC. In this study, we used adenoviral vector to deliver Bmi-1 shRNA driven by its own promoter to treat GC. Our results revealed that Ad-Bmi-1i could selectively silence Bmi-1 in GC cells which overexpress Bmi-1 and suppress the malignant phenotypes and stem-like properties of GC cells in vitro and in vivo. Moreover, direct injection of Ad-Bmi-1i into xenografts suppressed tumor growth and destroyed cancer cells in vivo. Ad-Bmi-1i inhibited the proliferation of GC cells mainly via inducing senescence in vitro, but it suppressed tumor through inducing senescence and apoptosis, and inhibiting angiogenesis in vivo. Bmi-1 knockdown by Ad-Bmi-1i downregulated VEGF via inhibiting AKT activity. These results suggest that Ad-Bmi-1i not only inhibits tumor growth and stem cell-like phenotype by inducing cellular senescence directly, but also has an indirect anti-tumor activity by anti-angiogenesis effects via regulating PTEN/AKT/VEGF pathway. Transfer of gene interference guided by its own promoter by an adeno-associated virus (AAV) vector might be a potent antitumor approach for cancer therapy. PMID:27009837

Characterizing Human Stem Cell–derived Sensory Neurons at the Single-cell Level Reveals Their Ion Channel Expression and Utility in Pain Research

PubMed Central

Young, Gareth T; Gutteridge, Alex; Fox, Heather DE; Wilbrey, Anna L; Cao, Lishuang; Cho, Lily T; Brown, Adam R; Benn, Caroline L; Kammonen, Laura R; Friedman, Julia H; Bictash, Magda; Whiting, Paul; Bilsland, James G; Stevens, Edward B

2014-01-01

The generation of human sensory neurons by directed differentiation of pluripotent stem cells opens new opportunities for investigating the biology of pain. The inability to generate this cell type has meant that up until now their study has been reliant on the use of rodent models. Here, we use a combination of population and single-cell techniques to perform a detailed molecular, electrophysiological, and pharmacological phenotyping of sensory neurons derived from human embryonic stem cells. We describe the evolution of cell populations over 6 weeks of directed differentiation; a process that results in the generation of a largely homogeneous population of neurons that are both molecularly and functionally comparable to human sensory neurons derived from mature dorsal root ganglia. This work opens the prospect of using pluripotent stem-cell–derived sensory neurons to study human neuronal physiology and as in vitro models for drug discovery in pain and sensory disorders. PMID:24832007

Cell of Origin and Cancer Stem Cell Phenotype in Medulloblastomas

DTIC Science & Technology

2017-09-01

AWARD NUMBER: W81XWH-14-1-0115 TITLE: Cell of Origin and Cancer Stem Cell Phenotype in Medulloblastomas PRINCIPAL INVESTIGATOR: Kyuson Yun...CA130273 - Cell of Origin and Cancer Stem Cell Phenotype in Medulloblastomas 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0115 5c. PROGRAM...hypothesis, we originally proposed to transform neural stem cells (NSCs) and neural progenitor cells (NPCs) in vivo by expressing an activated form

Adult subependymal neural precursors, but not differentiated cells, undergo rapid cathodal migration in the presence of direct current electric fields.

PubMed

Babona-Pilipos, Robart; Droujinine, Ilia A; Popovic, Milos R; Morshead, Cindi M

2011-01-01

The existence of neural stem and progenitor cells (together termed neural precursor cells) in the adult mammalian brain has sparked great interest in utilizing these cells for regenerative medicine strategies. Endogenous neural precursors within the adult forebrain subependyma can be activated following injury, resulting in their proliferation and migration toward lesion sites where they differentiate into neural cells. The administration of growth factors and immunomodulatory agents following injury augments this activation and has been shown to result in behavioural functional recovery following stroke. With the goal of enhancing neural precursor migration to facilitate the repair process we report that externally applied direct current electric fields induce rapid and directed cathodal migration of pure populations of undifferentiated adult subependyma-derived neural precursors. Using time-lapse imaging microscopy in vitro we performed an extensive single-cell kinematic analysis demonstrating that this galvanotactic phenomenon is a feature of undifferentiated precursors, and not differentiated phenotypes. Moreover, we have shown that the migratory response of the neural precursors is a direct effect of the electric field and not due to chemotactic gradients. We also identified that epidermal growth factor receptor (EGFR) signaling plays a role in the galvanotactic response as blocking EGFR significantly attenuates the migratory behaviour. These findings suggest direct current electric fields may be implemented in endogenous repair paradigms to promote migration and tissue repair following neurotrauma.

3-Deazaneplanocin A suppresses aggressive phenotype-related gene expression in an oral squamous cell carcinoma cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hatta, Mitsutoki, E-mail: hatta@college.fdcnet.ac.jp; Naganuma, Kaori; Kato, Kenichi

In tumor tissues, alterations of gene expression caused by aberrant epigenetic modifications confer phenotypic diversity on malignant cells. Although 3-deazaneplanocin A (DZNep) has been shown to reactivate tumor suppressor genes in several cancer cells, it remains unclear whether DZNep attenuates the malignant phenotypes of oral squamous cell carcinoma (OSCC) cells. In this study, we investigated the effect of DZNep on the expression of genes related to aggressive phenotypes, such as epithelial–mesenchymal transition, in OSCC cells. We found that DZNep reduced the cellular levels of polycomb group proteins (EZH2, SUZ12, BMI1, and RING1A) and the associated trimethylation of Lys27 on histonemore » H3 and monoubiquitination of Lys119 on histone H2A in the poorly differentiated OSCC cell line SAS. Immunocytochemical staining demonstrated that DZNep induced the reorganization of filamentous actin and the membrane localization of E-cadherin associated with cell–cell adhesions. We also found an inhibitory effect of DZNep on cell proliferation using a WST assay. Finally, quantitative RT-PCR analysis demonstrated that genes involved in the aggressive phenotypes (TWIST2, EGFR, ACTA2, TGFB1, WNT5B, and APLIN) were down-regulated, whereas epithelial phenotype genes (CDH1, CLDN4, IVL, and TGM1) were up-regulated in SAS cells treated with DZNep. Collectively, our findings suggest that DZNep reverses the aggressive characteristics of OSCC cells through the dynamic regulation of epithelial plasticity via the reprogramming of gene expression patterns. - Highlights: • DZNep reduced PcG proteins and associated histone modifications in OSCC cells. • DZNep enhanced cell–cell adhesion indicative of epithelial phenotype in OSCC cells. • DZNep suppressed the aggressive phenotype-related gene expression in OSCC cells. • DZNep activated the gene expression of epithelial markers in OSCC cells.« less

Caenorhabditis elegans anillin (ani-1) regulates neuroblast cytokinesis and epidermal morphogenesis during embryonic development.

PubMed

Fotopoulos, N; Wernike, D; Chen, Y; Makil, N; Marte, A; Piekny, A

2013-11-01

The formation of tissues is essential for metazoan development. During Caenorhabditis elegans embryogenesis, ventral epidermal cells migrate to encase the ventral surface of the embryo in a layer of epidermis by a process known as ventral enclosure. This process is regulated by guidance cues secreted by the underlying neuroblasts. However, since the cues and their receptors are differentially expressed in multiple cell types, the role of the neuroblasts in ventral enclosure is not fully understood. Furthermore, although F-actin is required for epidermal cell migration, it is not known if nonmuscle myosin is also required. Anillin (ANI-1) is an actin and myosin-binding protein that coordinates actin-myosin contractility in the early embryo. Here, we show that ANI-1 localizes to the cleavage furrows of dividing neuroblasts during mid-embryogenesis and is required for their division. Embryos depleted of ani-1 display a range of ventral enclosure phenotypes, where ventral epidermal cells migrate with similar speeds to control embryos, but contralateral neighbors often fail to meet and are misaligned. The ventral enclosure phenotypes in ani-1 RNAi embryos suggest that the position or shape of neuroblasts is important for directing ventral epidermal cell migration, although does not rule out an autonomous requirement for ani-1 in the epidermal cells. Furthermore, we show that rho-1 and other regulators of nonmuscle myosin activity are required for ventral epidermal cell migration. Interestingly, altering nonmuscle myosin contractility alleviates or strengthens ani-1's ventral enclosure phenotypes. Our findings suggest that ventral enclosure is a complex process that likely relies on inputs from multiple tissues. © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

EphA2 and ephrin-A5 are not a receptor-ligand pair in the ocular lens.

PubMed

Cheng, Catherine; Fowler, Velia M; Gong, Xiaohua

2017-09-01

Eph-ephrin bidirectional signaling is essential for eye lens transparency in humans and mice. Our previous studies in mouse lenses demonstrate that ephrin-A5 is mainly expressed in the anterior epithelium, where it is required for maintaining the anterior epithelial monolayer. In contrast, EphA2 is localized in equatorial epithelial and fiber cells where it is essential for equatorial epithelial and fiber cell organization and hexagonal cell shape. Immunostaining of lens epithelial and fiber cells reveals that EphA2 and ephrin-A5 are also co-expressed in anterior fiber cell tips, equatorial epithelial cells and newly formed lens fibers, although they are not precisely colocalized. Due to this complex expression pattern and the promiscuous interactions between Eph receptors and ephrin ligands, as well as their complex bidirectional signaling pathways, cataracts in ephrin-A5(-/-) or EphA2(-/-) lenses may arise from loss of function or abnormal signaling mechanisms. To test whether abnormal signaling mechanisms may play a role in cataractogenesis in ephrin-A5(-/-) or EphA2(-/-) lenses, we generated EphA2 and ephrin-A5 double knockout (DKO) mice. We compared the phenotypes of EphA2(-/-) and ephrin-A5(-/-) lenses to that of DKO lenses. DKO lenses displayed an additive lens phenotype that was not significantly different from the two single KO lens phenotypes. Similar to ephrin-A5(-/-) lenses, DKO lenses had abnormal anterior epithelial cells leading to a large mass of epithelial cells that invade into the underlying fiber cell layer, directly resulting in anterior cataracts in ephrin-A5(-/-) and DKO lenses. Yet, similar to EphA2(-/-) lenses, DKO lenses also had abnormal packing of equatorial epithelial cells with disorganized meridional rows, lack of a lens fulcrum and disrupted fiber cells. The DKO lens phenotype rules out abnormal signaling by EphA2 in ephrin-A5(-/-) lenses or by ephrin-A5 in EphA2(-/-) lenses as possible cataract mechanisms. Thus, these results indicate that EphA2 and ephrin-A5 do not form a lens receptor-ligand pair, and that EphA2 and ephrin-A5 have other binding partners in the lens to help align differentiating equatorial epithelial cells or maintain the anterior epithelium, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mucosal BCG Vaccination Induces Protective Lung-Resident Memory T Cell Populations against Tuberculosis.

PubMed

Perdomo, Carolina; Zedler, Ulrike; Kühl, Anja A; Lozza, Laura; Saikali, Philippe; Sander, Leif E; Vogelzang, Alexis; Kaufmann, Stefan H E; Kupz, Andreas

2016-11-22

Mycobacterium bovis Bacille Calmette-Guérin (BCG) is the only licensed vaccine against tuberculosis (TB), yet its moderate efficacy against pulmonary TB calls for improved vaccination strategies. Mucosal BCG vaccination generates superior protection against TB in animal models; however, the mechanisms of protection remain elusive. Tissue-resident memory T (T RM ) cells have been implicated in protective immune responses against viral infections, but the role of T RM cells following mycobacterial infection is unknown. Using a mouse model of TB, we compared protection and lung cellular infiltrates of parenteral and mucosal BCG vaccination. Adoptive transfer and gene expression analyses of lung airway cells were performed to determine the protective capacities and phenotypes of different memory T cell subsets. In comparison to subcutaneous vaccination, intratracheal and intranasal BCG vaccination generated T effector memory and T RM cells in the lung, as defined by surface marker phenotype. Adoptive mucosal transfer of these airway-resident memory T cells into naive mice mediated protection against TB. Whereas airway-resident memory CD4 + T cells displayed a mixture of effector and regulatory phenotype, airway-resident memory CD8 + T cells displayed prototypical T RM features. Our data demonstrate a key role for mucosal vaccination-induced airway-resident T cells in the host defense against pulmonary TB. These results have direct implications for the design of refined vaccination strategies. BCG remains the only licensed vaccine against TB. Parenterally administered BCG has variable efficacy against pulmonary TB, and thus, improved prevention strategies and a more refined understanding of correlates of vaccine protection are required. Induction of memory T cells has been shown to be essential for protective TB vaccines. Mimicking the natural infection route by mucosal vaccination has been known to generate superior protection against TB in animal models; however, the mechanisms of protection have remained elusive. Here we performed an in-depth analysis to dissect the immunological mechanisms associated with superior mucosal protection in the mouse model of TB. We found that mucosal, and not subcutaneous, BCG vaccination generates lung-resident memory T cell populations that confer protection against pulmonary TB. We establish a comprehensive phenotypic characterization of these populations, providing a framework for future vaccine development. Copyright © 2016 Perdomo et al.

Image-guided genomics of phenotypically heterogeneous populations reveals vascular signalling during symbiotic collective cancer invasion

PubMed Central

Konen, J.; Summerbell, E.; Dwivedi, B.; Galior, K.; Hou, Y.; Rusnak, L.; Chen, A.; Saltz, J.; Zhou, W.; Boise, L. H.; Vertino, P.; Cooper, L.; Salaita, K.; Kowalski, J.; Marcus, A. I.

2017-01-01

Phenotypic heterogeneity is widely observed in cancer cell populations. Here, to probe this heterogeneity, we developed an image-guided genomics technique termed spatiotemporal genomic and cellular analysis (SaGA) that allows for precise selection and amplification of living and rare cells. SaGA was used on collectively invading 3D cancer cell packs to create purified leader and follower cell lines. The leader cell cultures are phenotypically stable and highly invasive in contrast to follower cultures, which show phenotypic plasticity over time and minimally invade in a sheet-like pattern. Genomic and molecular interrogation reveals an atypical VEGF-based vasculogenesis signalling that facilitates recruitment of follower cells but not for leader cell motility itself, which instead utilizes focal adhesion kinase-fibronectin signalling. While leader cells provide an escape mechanism for followers, follower cells in turn provide leaders with increased growth and survival. These data support a symbiotic model of collective invasion where phenotypically distinct cell types cooperate to promote their escape. PMID:28497793

Phenotypic Plasticity and Cell Fate Decisions in Cancer: Insights from Dynamical Systems Theory

PubMed Central

Kulkarni, Prakash; Levine, Herbert

2017-01-01

Waddington’s epigenetic landscape, a famous metaphor in developmental biology, depicts how a stem cell progresses from an undifferentiated phenotype to a differentiated one. The concept of “landscape” in the context of dynamical systems theory represents a high-dimensional space, in which each cell phenotype is considered as an “attractor” that is determined by interactions between multiple molecular players, and is buffered against environmental fluctuations. In addition, biological noise is thought to play an important role during these cell-fate decisions and in fact controls transitions between different phenotypes. Here, we discuss the phenotypic transitions in cancer from a dynamical systems perspective and invoke the concept of “cancer attractors”—hidden stable states of the underlying regulatory network that are not occupied by normal cells. Phenotypic transitions in cancer occur at varying levels depending on the context. Using epithelial-to-mesenchymal transition (EMT), cancer stem-like properties, metabolic reprogramming and the emergence of therapy resistance as examples, we illustrate how phenotypic plasticity in cancer cells enables them to acquire hybrid phenotypes (such as hybrid epithelial/mesenchymal and hybrid metabolic phenotypes) that tend to be more aggressive and notoriously resilient to therapies such as chemotherapy and androgen-deprivation therapy. Furthermore, we highlight multiple factors that may give rise to phenotypic plasticity in cancer cells, such as (a) multi-stability or oscillatory behaviors governed by underlying regulatory networks involved in cell-fate decisions in cancer cells, and (b) network rewiring due to conformational dynamics of intrinsically disordered proteins (IDPs) that are highly enriched in cancer cells. We conclude by discussing why a therapeutic approach that promotes “recanalization”, i.e., the exit from “cancer attractors” and re-entry into “normal attractors”, is more likely to succeed rather than a conventional approach that targets individual molecules/pathways. PMID:28640191

Manufacturing Cell Therapies Using Engineered Biomaterials.

PubMed

Abdeen, Amr A; Saha, Krishanu

2017-10-01

Emerging manufacturing processes to generate regenerative advanced therapies can involve extensive genomic and/or epigenomic manipulation of autologous or allogeneic cells. These cell engineering processes need to be carefully controlled and standardized to maximize safety and efficacy in clinical trials. Engineered biomaterials with smart and tunable properties offer an intriguing tool to provide or deliver cues to retain stemness, direct differentiation, promote reprogramming, manipulate the genome, or select functional phenotypes. This review discusses the use of engineered biomaterials to control human cell manufacturing. Future work exploiting engineered biomaterials has the potential to generate manufacturing processes that produce standardized cells with well-defined critical quality attributes appropriate for clinical testing. Copyright © 2017 Elsevier Ltd. All rights reserved.

Collective Motion in Bacterial Populations with Mixed Phenotypic Behaviors

NASA Astrophysics Data System (ADS)

Hoeger, Kentaro; Strickland, Ben; Shoup, Daniel; Ursell, Tristan

The motion of large, densely packed groups of organisms is often qualitatively distinct from the motion of individuals, yet hinges on individual properties and behaviors. Collective motion of bacteria depends strongly on the phenotypic behaviors of individual cells, the physical interactions between cells, and the geometry of their environment, often with multiple phenotypes coexisting in a population. Thus, to characterize how these selectively important interactions affect group traits, such as cell dispersal, spatial segregation of phenotypes, and material transport in groups, we use a library of Bacillus subtilis mutants that modulate chemotaxis, motility, and biofilm formation. By mixing phenotypes and observing bacterial behaviors and motion at single cell resolution, we probe collective motion as a function of phenotypic mixture and environmental geometry. Our work demonstrates that collective microbial motion exhibits a transition, from `turbulence' to semiballistic burrowing, as phenotypic composition varies. This work illuminates the role that individual cell behaviors play in the emergence of collective motion, and may signal qualitatively distinct regimes of material transport in bacterial populations. University of Oregon.

CD4 Receptor is a Key Determinant of Divergent HIV-1 Sensing by Plasmacytoid Dendritic Cells

PubMed Central

Wilen, Craig; Gopal, Ramya; Huq, Rumana; Wu, Vernon; Sunseri, Nicole; Bhardwaj, Nina

2016-01-01

Plasmacytoid dendritic cells (pDC) are innate immune cells that sense viral nucleic acids through endosomal Toll-like receptor (TLR) 7/9 to produce type I interferon (IFN) and to differentiate into potent antigen presenting cells (APC). Engagement of TLR7/9 in early endosomes appears to trigger the IRF7 pathway for IFN production whereas engagement in lysosomes seems to trigger the NF-κB pathway for maturation into APC. We showed previously that HIV-1 (HIV) localizes predominantly to early endosomes, not lysosomes, and mainly stimulate IRF7 rather than NF-κB signaling pathways in pDC. This divergent signaling may contribute to disease progression through production of pro-apoptotic and pro-inflammatory IFN and inadequate maturation of pDCs. We now demonstrate that HIV virions may be re-directed to lysosomes for NF-κB signaling by either pseudotyping HIV with influenza hemagglutinin envelope or modification of CD4 mediated-intracellular trafficking. These data suggest that HIV envelope-CD4 receptor interactions drive pDC activation toward an immature IFN producing phenotype rather than differentiation into a mature dendritic cell phenotype. PMID:27082754

Metabolic phenotyping of a model of adipocyte differentiation

PubMed Central

Roberts, Lee D.; Virtue, Sam; Vidal-Puig, Antonio; Nicholls, Andrew W.

2009-01-01

The 3T3-L1 murine cell line is a robust and widely used model for the study of adipogenesis and processes occurring in mature adipocytes. The fibroblastic like cells can be induced by hormones to differentiate into mature adipocytes. In this study, the metabolic phenotype associated with differentiation of the 3T3-L1 cell line has been studied using gas chromatography-mass spectrometry, 1H nuclear magnetic resonance spectroscopy, liquid chromatography-mass spectrometry, direct infusion-mass spectrometry, and 13C substrate labeling in conjunction with multivariate statistics. The changes in metabolite concentrations at distinct periods during differentiation have been defined including alterations in the TCA cycle, glycolysis, the production of odd chain fatty acids by α-oxidation, fatty acid synthesis, fatty acid desaturation, polyamine biosynthesis, and trans-esterification to produce complex lipids. The metabolic changes induced during differentiation of the 3T3-L1 cell line were then compared with the metabolic differences between pre- and postdifferentiation primary adipocytes. These metabolic alterations reflect the changing role of the 3T3-L1 cells during differentiation, as well as possibly providing metabolic triggers to stimulate the processes which occur during differentiation. PMID:19602617

The impact of cellular senescence in skin ageing: A notion of mosaic and therapeutic strategies.

PubMed

Toutfaire, Marie; Bauwens, Emilie; Debacq-Chainiaux, Florence

2017-10-15

Cellular senescence is now recognized as one of the nine hallmarks of ageing. Recent data show the involvement of senescent cells in tissue ageing and some age-related diseases. Skin represents an ideal model for the study of ageing. Indeed, skin ageing varies between individuals depending on their chronological age but also on their exposure to various exogenous factors (mainly ultraviolet rays). If senescence traits can be detected with ageing in the skin, the senescent phenotype varies among the various skin cell types. Moreover, the origin of cellular senescence in the skin is still unknown, and multiple origins are possible. This reflects the mosaic of skin ageing. Senescent cells can interfere with their microenvironment, either via the direct secretion of factors (the senescence-associated secretory phenotype) or via other methods of communication, such as extracellular vesicles. Knowledge regarding the impact of cellular senescence on skin ageing could be integrated into dermatology research, especially to limit the appearance of senescent cells after photo(chemo)therapy or in age-related skin diseases. Therapeutic approaches include the clearance of senescent cells via the use of senolytics or via the cooperation with the immune system. Copyright © 2017 Elsevier Inc. All rights reserved.

A high-content image-based method for quantitatively studying context-dependent cell population dynamics

PubMed Central

Garvey, Colleen M.; Spiller, Erin; Lindsay, Danika; Chiang, Chun-Te; Choi, Nathan C.; Agus, David B.; Mallick, Parag; Foo, Jasmine; Mumenthaler, Shannon M.

2016-01-01

Tumor progression results from a complex interplay between cellular heterogeneity, treatment response, microenvironment and heterocellular interactions. Existing approaches to characterize this interplay suffer from an inability to distinguish between multiple cell types, often lack environmental context, and are unable to perform multiplex phenotypic profiling of cell populations. Here we present a high-throughput platform for characterizing, with single-cell resolution, the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations both during standard growth and in response to multiple, co-occurring selective pressures. The speed of this platform enables a thorough investigation of the impacts of diverse selective pressures including genetic alterations, therapeutic interventions, heterocellular components and microenvironmental factors. The platform has been applied to both 2D and 3D culture systems and readily distinguishes between (1) cytotoxic versus cytostatic cellular responses; and (2) changes in morphological features over time and in response to perturbation. These important features can directly influence tumor evolution and clinical outcome. Our image-based approach provides a deeper insight into the cellular dynamics and heterogeneity of tumors (or other complex systems), with reduced reagents and time, offering advantages over traditional biological assays. PMID:27452732

A high-content image-based method for quantitatively studying context-dependent cell population dynamics

NASA Astrophysics Data System (ADS)

Garvey, Colleen M.; Spiller, Erin; Lindsay, Danika; Chiang, Chun-Te; Choi, Nathan C.; Agus, David B.; Mallick, Parag; Foo, Jasmine; Mumenthaler, Shannon M.

2016-07-01

Tumor progression results from a complex interplay between cellular heterogeneity, treatment response, microenvironment and heterocellular interactions. Existing approaches to characterize this interplay suffer from an inability to distinguish between multiple cell types, often lack environmental context, and are unable to perform multiplex phenotypic profiling of cell populations. Here we present a high-throughput platform for characterizing, with single-cell resolution, the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations both during standard growth and in response to multiple, co-occurring selective pressures. The speed of this platform enables a thorough investigation of the impacts of diverse selective pressures including genetic alterations, therapeutic interventions, heterocellular components and microenvironmental factors. The platform has been applied to both 2D and 3D culture systems and readily distinguishes between (1) cytotoxic versus cytostatic cellular responses; and (2) changes in morphological features over time and in response to perturbation. These important features can directly influence tumor evolution and clinical outcome. Our image-based approach provides a deeper insight into the cellular dynamics and heterogeneity of tumors (or other complex systems), with reduced reagents and time, offering advantages over traditional biological assays.

MiR-29b Downregulation Induces Phenotypic Modulation of Vascular Smooth Muscle Cells: Implication for Intracranial Aneurysm Formation and Progression to Rupture.

PubMed

Sun, Liqian; Zhao, Manman; Zhang, Jingbo; Lv, Ming; Li, Youxiang; Yang, Xinjian; Liu, Aihua; Wu, Zhongxue

2017-01-01

Our previous microarray results identified numerous microRNAs (miRNAs), including miR-29b, that were differentially expressed in the serum of intracranial aneurysm (IA) patients. The current study aimed to investigate whether miR-29b downregulation in IA could promote the phenotypic modulation of vascular smooth muscle cells (VSMCs) involved in the pathogenesis of aneurysm by activating ATG14-mediated autophagy. First, the levels of miR-29b and autophagy related genes (ATGs) between IA patients and normal subjects were compared. Next, we modified the level of miR-29b via lentivirus particles in the VSMCs and examined the effects of miR-29b on proliferation, migration, and phenotypic modulation of VSMCs from a contractile phenotype to a synthetic phenotype, as well as the levels of autophagy. Finally, the binding of miR-29b to the 3'UTR of ATG14 mRNA and its effects on ATG14 expression were analysed by a luciferase reporter assay and Western blot, respectively. The level of miR-29b was decreased, and autophagy markers were increased in the IA patients compared to that of the normal subjects. Knockdown of miR-29b significantly promoted VSMCs proliferation and migration and, more importantly, induced the phenotypic modulation associated with autophagy activation, whereas miR-29b overexpression showed the opposite effects. The luciferase reporter assay demonstrated that ATG14 was a functional target gene of miR-29b. Notably, knockdown of ATG14 by siRNA apparently abrogated miR-29b inhibition-mediated phenotypic modulation. Downregulation of miR-29b induced VSMCs phenotypic modulation by directly activating ATG14-mediated autophagy, which is associated with the formation, growth and rupture of IAs. © 2017 The Author(s) Published by S. Karger AG, Basel.

MEF2C-MYOCD and Leiomodin1 Suppression by miRNA-214 Promotes Smooth Muscle Cell Phenotype Switching in Pulmonary Arterial Hypertension

PubMed Central

Sahoo, Sanghamitra; Meijles, Daniel N.; Al Ghouleh, Imad; Tandon, Manuj; Cifuentes-Pagano, Eugenia; Sembrat, John; Rojas, Mauricio; Goncharova, Elena; Pagano, Patrick J.

2016-01-01

Background Vascular hyperproliferative disorders are characterized by excessive smooth muscle cell (SMC) proliferation leading to vessel remodeling and occlusion. In pulmonary arterial hypertension (PAH), SMC phenotype switching from a terminally differentiated contractile to synthetic state is gaining traction as our understanding of the disease progression improves. While maintenance of SMC contractile phenotype is reportedly orchestrated by a MEF2C-myocardin (MYOCD) interplay, little is known regarding molecular control at this nexus. Moreover, the burgeoning interest in microRNAs (miRs) provides the basis for exploring their modulation of MEF2C-MYOCD signaling, and in turn, a pro-proliferative, synthetic SMC phenotype. We hypothesized that suppression of SMC contractile phenotype in pulmonary hypertension is mediated by miR-214 via repression of the MEF2C-MYOCD-leiomodin1 (LMOD1) signaling axis. Methods and Results In SMCs isolated from a PAH patient cohort and commercially obtained hPASMCs exposed to hypoxia, miR-214 expression was monitored by qRT-PCR. miR-214 was upregulated in PAH- vs. control subject hPASMCs as well as in commercially obtained hPASMCs exposed to hypoxia. These increases in miR-214 were paralleled by MEF2C, MYOCD and SMC contractile protein downregulation. Of these, LMOD1 and MEF2C were directly targeted by the miR. Mir-214 overexpression mimicked the PAH profile, downregulating MEF2C and LMOD1. AntagomiR-214 abrogated hypoxia-induced suppression of the contractile phenotype and its attendant proliferation. Anti-miR-214 also restored PAH-PASMCs to a contractile phenotype seen during vascular homeostasis. Conclusions Our findings illustrate a key role for miR-214 in modulation of MEF2C-MYOCD-LMOD1 signaling and suggest that an antagonist of miR-214 could mitigate SMC phenotype changes and proliferation in vascular hyperproliferative disorders including PAH. PMID:27144530

Expression of c-Fes protein isoforms correlates with differentiation in myeloid leukemias.

PubMed

Carlson, Anne; Berkowitz, Jeanne McAdara; Browning, Damaris; Slamon, Dennis J; Gasson, Judith C; Yates, Karen E

2005-05-01

The cellular fes gene encodes a 93-kilodalton protein-tyrosine kinase (p93) that is expressed in both normal and neoplastic myeloid cells. Increased c-Fes expression is associated with differentiation in normal myeloid cells and cell lines. Our hypothesis was that primary leukemia cells would show a similar pattern of increased expression in more differentiated cells. Therefore, we compared c-Fes expression in cells with an undifferentiated, blast phenotype (acute myelogenous leukemia--AML) to cells with a differentiated phenotype (chronic myelogenous leukemia--CML). Instead of differences in p93 expression levels, we found complex patterns of c-Fes immunoreactive proteins that corresponded with differentiation in normal and leukemic myeloid cells. The "blast" pattern consisted of c-Fes immunoreactive proteins p93, p74, and p70; the "differentiated" pattern showed two additional c-Fes immunoreactive proteins, p67 and p62. Using mRNA from mouse and human cell lines, we found deletion of one or more exons in the c-fes mRNA. Those deletions predicted truncation of conserved domains (CDC15/FCH and SH2) involved in protein-protein interactions. No deletions were found, however, within the kinase domain. We infer that alternative splicing generates a family of c-Fes proteins. This may be a mechanism to direct the c-Fes kinase domain to different subcellular locations and/or substrates at specific stages of myeloid cell differentiation.

Micropatterned mammalian cells exhibit phenotype-specific left-right asymmetry.

PubMed

Wan, Leo Q; Ronaldson, Kacey; Park, Miri; Taylor, Grace; Zhang, Yue; Gimble, Jeffrey M; Vunjak-Novakovic, Gordana

2011-07-26

Left-right (LR) asymmetry (handedness, chirality) is a well-conserved biological property of critical importance to normal development. Changes in orientation of the LR axis due to genetic or environmental factors can lead to malformations and disease. While the LR asymmetry of organs and whole organisms has been extensively studied, little is known about the LR asymmetry at cellular and multicellular levels. Here we show that the cultivation of cell populations on micropatterns with defined boundaries reveals intrinsic cell chirality that can be readily determined by image analysis of cell alignment and directional motion. By patterning 11 different types of cells on ring-shaped micropatterns of various sizes, we found that each cell type exhibited definite LR asymmetry (p value down to 10(-185)) that was different between normal and cancer cells of the same type, and not dependent on surface chemistry, protein coating, or the orientation of the gravitational field. Interestingly, drugs interfering with actin but not microtubule function reversed the LR asymmetry in some cell types. Our results show that micropatterned cell populations exhibit phenotype-specific LR asymmetry that is dependent on the functionality of the actin cytoskeleton. We propose that micropatterning could potentially be used as an effective in vitro tool to study the initiation of LR asymmetry in cell populations, to diagnose disease, and to study factors involved with birth defects in laterality.

Culture on 3D Chitosan-Hyaluronic Acid Scaffolds Enhances Stem Cell Marker Expression and Drug Resistance in Human Glioblastoma Cancer Stem Cells.

PubMed

Wang, Kui; Kievit, Forrest M; Erickson, Ariane E; Silber, John R; Ellenbogen, Richard G; Zhang, Miqin

2016-12-01

The lack of in vitro models that support the growth of glioblastoma (GBM) stem cells (GSCs) that underlie clinical aggressiveness hinders developing new, effective therapies for GBM. While orthotopic patient-derived xenograft models of GBM best reflect in vivo tumor behavior, establishing xenografts is a time consuming, costly, and frequently unsuccessful endeavor. To address these limitations, a 3D porous scaffold composed of chitosan and hyaluronic acid (CHA) is synthesized. Growth and expression of the cancer stem cell (CSC) phenotype of the GSC GBM6 taken directly from fresh xenogratfs grown on scaffolds or as adherent monolayers is compared. While 2D adherent cultures grow as monolayers of flat epitheliod cells, GBM6 cells proliferate within pores of CHA scaffolds as clusters of self-adherent ovoid cells. Growth on scaffolds is accompanied by greater expression of genes that mediate epithelial-mesenchymal transition and maintain a primitive, undifferentiated phenotype, hallmarks of CSCs. Scaffold-grown cells also display higher expression of genes that promote resistance to hypoxia-induced oxidative stress. In accord, scaffold-grown cells show markedly greater resistance to clinically utilized alkylating agents compared to adherent cells. These findings suggest that our CHA scaffolds better mimic in vivo biological and clinical behavior and provide insights for developing novel individualized treatments. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative Temporal in Vivo Proteomics Deciphers the Transition of Virus-Driven Myeloid Cells into M2 Macrophages

PubMed Central

2017-01-01

Myeloid cells play a central role in the context of viral eradication, yet precisely how these cells differentiate throughout the course of acute infections is poorly understood. In this study, we have developed a novel quantitative temporal in vivo proteomics (QTiPs) platform to capture proteomic signatures of temporally transitioning virus-driven myeloid cells directly in situ, thus taking into consideration host–virus interactions throughout the course of an infection. QTiPs, in combination with phenotypic, functional, and metabolic analyses, elucidated a pivotal role for inflammatory CD11b+, Ly6G–, Ly6Chigh-low cells in antiviral immune response and viral clearance. Most importantly, the time-resolved QTiPs data set showed the transition of CD11b+, Ly6G–, Ly6Chigh-low cells into M2-like macrophages, which displayed increased antigen-presentation capacities and bioenergetic demands late in infection. We elucidated the pivotal role of myeloid cells in virus clearance and show how these cells phenotypically, functionally, and metabolically undergo a timely transition from inflammatory to M2-like macrophages in vivo. With respect to the growing appreciation for in vivo examination of viral–host interactions and for the role of myeloid cells, this study elucidates the use of quantitative proteomics to reveal the role and response of distinct immune cell populations throughout the course of virus infection. PMID:28768414

Epigenetic control of CD8+ T cell differentiation.

PubMed

Henning, Amanda N; Roychoudhuri, Rahul; Restifo, Nicholas P

2018-05-01

Upon stimulation, small numbers of naive CD8 + T cells proliferate and differentiate into a variety of memory and effector cell types. CD8 + T cells can persist for years and kill tumour cells and virally infected cells. The functional and phenotypic changes that occur during CD8 + T cell differentiation are well characterized, but the epigenetic states that underlie these changes are incompletely understood. Here, we review the epigenetic processes that direct CD8 + T cell differentiation and function. We focus on epigenetic modification of DNA and associated histones at genes and their regulatory elements. We also describe structural changes in chromatin organization that affect gene expression. Finally, we examine the translational potential of epigenetic interventions to improve CD8 + T cell function in individuals with chronic infections and cancer.

Models of GH deficiency in animal studies.

PubMed

Gahete, Manuel D; Luque, Raul M; Castaño, Justo P

2016-12-01

Growth hormone (GH) is a peptide hormone released from pituitary somatotrope cells that promotes growth, cell division and regeneration by acting directly through the GH receptor (GHR), or indirectly via hepatic insulin-like growth factor 1 (IGF1) production. GH deficiency (GHD) can cause severe consequences, such as growth failure, changes in body composition and altered insulin sensitivity, depending of the origin, time of onset (childhood or adulthood) or duration of GHD. The highly variable clinical phenotypes of GHD can now be better understood through research on transgenic and naturally-occurring animal models, which are widely employed to investigate the origin, phenotype, and consequences of GHD, and particularly the underlying mechanisms of metabolic disorders associated to GHD. Here, we reviewed the most salient aspects of GH biology, from somatotrope development to GH actions, linked to certain GHD types, as well as the animal models employed to reproduce these GHD-associated alterations. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cytokeratin characterization of human prostatic carcinoma and its derived cell lines.

PubMed

Nagle, R B; Ahmann, F R; McDaniel, K M; Paquin, M L; Clark, V A; Celniker, A

1987-01-01

Two murine monoclonal anti-cytokeratin antibodies with defined specificity were shown to distinguish between basal cells and luminal cells in human prostate tissue. Forty-one biopsies or transurethral resection specimens were characterized using these two antibodies. In cases of benign prostatic hyperplasia, focal loss of the basal cell layer was noted in areas of glandular proliferation. Ten cases of adenocarcinoma of the prostate, varying in Gleason's histological grade from 2 to 4, were also studied. In each case the carcinoma was shown to represent the luminal cell phenotype with no evidence of involvement of the basal cell phenotype. An analysis of three established metastatic prostatic carcinoma cell lines (DU-145, PC-3, and LNCaP) using two-dimensional electrophoresis showed that the cytokeratin complement of each cell line was slightly different but retained the phenotype of the luminal cell. It was concluded that during both hyperplasia and neoplastic transformation of the prostate, the luminal cell phenotype is primarily involved and that the basal cell phenotype does not appear to contribute to either intraluminal proliferation or invasive cell populations.

Poly(ADP-ribose) polymerase-1 regulates microglia mediated decrease of endothelial tight junction integrity.

PubMed

Mehrabadi, Abbas Rezaeian; Korolainen, Minna A; Odero, Gary; Miller, Donald W; Kauppinen, Tiina M

2017-09-01

Alzheimer's disease pathology includes, beside neuronal damage, reactive gliosis and reduced blood-brain barrier (BBB) integrity. Microglia are intimately associated with the BBB and upon AD pathology, pro-inflammatory responses of microglia could contribute to BBB damage. To study whether microglia can directly affect BBB integrity, the effects of amyloid beta (Aβ) -stimulated primary murine microglia on co-cultured mouse brain endothelial cells (bEnd3) and murine astrocyte cultures were assessed. We also assessed whether microglial phenotype modulation via poly(ADP-ribose) polymerase-1 (PARP-1) inhibition/ablation can reverse microglial impact on these BBB forming cells. Unstimulated microglia promoted expression of tight junction proteins (TJPs), zonula ocluden-1 (ZO-1) and occludin in co-cultured endothelia cells, whereas Aβ-stimulated microglia reduced endothelial expression of ZO-1 and occludin. Astrocytes co-cultured with microglia showed elevated glial fibrillary acidic protein (GFAP) expression, which was further increased if microglia had been stimulated with Aβ. Aβ induced microglial release of nitric oxide (NO) and tumour necrosis factor alpha (TNFα), which resulted in reduced endothelial expression of TJPs and increased paracellular permeability. Microglial PARP-1 inhibition attenuated these Aβ-induced events. These findings demonstrate that PARP-1 mediated microglial responses (NO and TNFα) can directly reduce BBB integrity by promoting TJP degradation, increasing endothelial cell permeability and inducing astrogliosis. PARP-1 as a modulator of microglial phenotype can prevent microglial BBB damaging events, and thus is a potential therapeutic target. Copyright © 2017 Elsevier Ltd. All rights reserved.

Programming cells by multiplex genome engineering and accelerated evolution.

PubMed

Wang, Harris H; Isaacs, Farren J; Carr, Peter A; Sun, Zachary Z; Xu, George; Forest, Craig R; Church, George M

2009-08-13

The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales. Although in vitro and directed evolution methods have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes. Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). We applied MAGE to optimize the 1-deoxy-D-xylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. We isolated variants with more than fivefold increase in lycopene production within 3 days, a significant improvement over existing metabolic engineering techniques. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.

Transcription factor-based modulation of neural stem cell differentiation using direct protein transduction

PubMed Central

Stock, Kristin; Nolden, Lars; Edenhofer, Frank; Quandel, Tamara

2010-01-01

In contrast to conventional gene transfer strategies, the direct introduction of recombinant proteins into cells bypasses the risk of insertional mutagenesis and offers an alternative to genetic intervention. Here, we explore whether protein transduction of the gliogenic transcription factor Nkx2.2 can be used to promote oligodendroglial differentiation of mouse embryonic stem cell (ESC)-derived neural stem cells (NSC). To that end, a recombinant cell-permeant form of Nkx2.2 protein was generated. Exposure of ESC-derived NSC to the recombinant protein and initiation of differentiation resulted in a two-fold increase in the number of oligodendrocytes. Furthermore, Nkx2.2-transduced cells exhibited a more mature oligodendroglial phenotype. Comparative viral gene transfer studies showed that the biological effect of Nkx2.2 protein transduction is comparable to that obtained by lentiviral transduction. The results of this proof-of-concept study depict direct intracellular delivery of transcription factors as alternative modality to control lineage differentiation in NSC cultures without genetic modification. Electronic supplementary material The online version of this article (doi:10.1007/s00018-010-0347-1) contains supplementary material, which is available to authorized users. PMID:20352468

Common genetic variation drives molecular heterogeneity in human iPSCs.

PubMed

Kilpinen, Helena; Goncalves, Angela; Leha, Andreas; Afzal, Vackar; Alasoo, Kaur; Ashford, Sofie; Bala, Sendu; Bensaddek, Dalila; Casale, Francesco Paolo; Culley, Oliver J; Danecek, Petr; Faulconbridge, Adam; Harrison, Peter W; Kathuria, Annie; McCarthy, Davis; McCarthy, Shane A; Meleckyte, Ruta; Memari, Yasin; Moens, Nathalie; Soares, Filipa; Mann, Alice; Streeter, Ian; Agu, Chukwuma A; Alderton, Alex; Nelson, Rachel; Harper, Sarah; Patel, Minal; White, Alistair; Patel, Sharad R; Clarke, Laura; Halai, Reena; Kirton, Christopher M; Kolb-Kokocinski, Anja; Beales, Philip; Birney, Ewan; Danovi, Davide; Lamond, Angus I; Ouwehand, Willem H; Vallier, Ludovic; Watt, Fiona M; Durbin, Richard; Stegle, Oliver; Gaffney, Daniel J

2017-06-15

Technology utilizing human induced pluripotent stem cells (iPS cells) has enormous potential to provide improved cellular models of human disease. However, variable genetic and phenotypic characterization of many existing iPS cell lines limits their potential use for research and therapy. Here we describe the systematic generation, genotyping and phenotyping of 711 iPS cell lines derived from 301 healthy individuals by the Human Induced Pluripotent Stem Cells Initiative. Our study outlines the major sources of genetic and phenotypic variation in iPS cells and establishes their suitability as models of complex human traits and cancer. Through genome-wide profiling we find that 5-46% of the variation in different iPS cell phenotypes, including differentiation capacity and cellular morphology, arises from differences between individuals. Additionally, we assess the phenotypic consequences of genomic copy-number alterations that are repeatedly observed in iPS cells. In addition, we present a comprehensive map of common regulatory variants affecting the transcriptome of human pluripotent cells.

New Directions for Natural Killer T Cells in the Immunotherapy of Cancer

PubMed Central

Teyton, Luc

2017-01-01

Natural killer T (NKT) cells have been placed at the interface between innate and adaptive immunity by a long series of experiments that convincingly showed that beyond cytokine secretion and NK cell recruitment, NKT cells were coordinating dendritic cell and B cell maturation through direct membrane contacts and initiate productive responses. As such, NKT cells are the cellular adjuvant of many immune reactions and have functions that go much beyond what their name encapsulates. In addition, the initial discovery of the ligands of NKT cells is deeply linked to cancer biology and therapy. However, for a host of reasons, animal models in which agonists of NKT cells were used did not translate well to human cancers. A systematic reassessment of NKT cells role in tumorigenesis, especially spontaneous one, is now accessible using single cell analysis technologies both in mouse and man, and should be taken advantage of. Similarly, the migration, localization, phenotype of NKT cells following induced expansion after injection of an agonist can be examined at the single cell level. This technological revolution will help evaluate where and how NKT cells can be used in cancer. PMID:29209309

Overexpression of molecular chaperons GRP78 and GRP94 in CD44(hi)/CD24(lo) breast cancer stem cells.

PubMed

Nami, Babak; Ghasemi-Dizgah, Armin; Vaseghi, Akbar

2016-01-01

Breast cancer stem cell with CD44(hi)/CD24(lo) phonotype is described having stem cell properties and represented as the main driving factor in breast cancer initiation, growth, metastasis and low response to anti-cancer agents. Glucoseregulated proteins (GRPs) are heat shock protein family chaperons that are charged with regulation of protein machinery and modulation of endoplasmic reticulum homeostasis whose important roles in stem cell development and invasion of various cancers have been demonstrated. Here, we investigated the expression levels of GRP78 and GRP94 in CD44(hi)/CD24(lo) phenotype breast cancer stem cells (BCSCs). MCF7, T-47D and MDA-MB-231 breast cancer cell lines were used. CD44(hi)/CD24(lo) phenotype cell population were analyzed and sorted by fluorescence-activated cell sorting (FACS). Transcriptional and translational expression of GRP78 and GRP94 were investigated by western blotting and quantitative real time PCR. RESULTS showed different proportion of CD44(hi)/CD24(lo) phenotype cell population in their original bulk cells. The ranking of the cell lines in terms of CD44(hi)/CD24(lo) phenotype cell population was as MCF7

Directional selection effects on patterns of phenotypic (co)variation in wild populations

PubMed Central

Patton, J. L.; Hubbe, A.; Marroig, G.

2016-01-01

Phenotypic (co)variation is a prerequisite for evolutionary change, and understanding how (co)variation evolves is of crucial importance to the biological sciences. Theoretical models predict that under directional selection, phenotypic (co)variation should evolve in step with the underlying adaptive landscape, increasing the degree of correlation among co-selected traits as well as the amount of genetic variance in the direction of selection. Whether either of these outcomes occurs in natural populations is an open question and thus an important gap in evolutionary theory. Here, we documented changes in the phenotypic (co)variation structure in two separate natural populations in each of two chipmunk species (Tamias alpinus and T. speciosus) undergoing directional selection. In populations where selection was strongest (those of T. alpinus), we observed changes, at least for one population, in phenotypic (co)variation that matched theoretical expectations, namely an increase of both phenotypic integration and (co)variance in the direction of selection and a re-alignment of the major axis of variation with the selection gradient. PMID:27881744

Survival signals and targets for therapy in breast implant-associated ALK--anaplastic large cell lymphoma.

PubMed

Lechner, Melissa G; Megiel, Carolina; Church, Connor H; Angell, Trevor E; Russell, Sarah M; Sevell, Rikki B; Jang, Julie K; Brody, Garry S; Epstein, Alan L

2012-09-01

Anaplastic lymphoma kinase (ALK)-negative, T-cell, anaplastic, non-Hodgkin lymphoma (T-ALCL) in patients with textured saline and silicone breast implants is a recently recognized clinical entity for which the etiology and optimal treatment remain unknown. Using three newly established model cell lines from patient biopsy specimens, designated T-cell breast lymphoma (TLBR)-1 to -3, we characterized the phenotype and function of these tumors to identify mechanisms of cell survival and potential therapeutic targets. Cytogenetics revealed chromosomal atypia with partial or complete trisomy and absence of the NPM-ALK (2;5) translocation. Phenotypic characterization showed strong positivity for CD30, CD71, T-cell CD2/5/7, and antigen presentation (HLA-DR, CD80, CD86) markers, and interleukin (IL)-2 (CD25, CD122) and IL-6 receptors. Studies of these model cell lines showed strong activation of STAT3 signaling, likely related to autocrine production of IL-6 and decreased SHP-1. STAT3 inhibition, directly or by recovery of SHP-1, and cyclophosphamide-Adriamycin-vincristine-prednisone (CHOP) chemotherapy reagents, effectively kill cells of all three TLBR models in vitro and may be pursued as therapies for patients with breast implant-associated T-ALCLs. The TLBR cell lines closely resemble the primary breast implant-associated lymphomas from which they were derived and as such provide valuable preclinical models to study their unique biology. ©2012 AACR.

Micro-composite substrates for the study of cell-matrix mechanical interactions.

PubMed

Chao, Pen-hsiu Grace; Sheng, Shou-Chien; Chang, Wei-Ren

2014-10-01

The chemical and physical gradients in the native cell microenvironment induce intracellular polarization and control cell behaviors such as morphology, migration and phenotypic changes. Directed cell migration in response to substrate stiffness gradients, known as durotaxis or mechanotaxis, has drawn attention due to its significance in development, metastasis, and wound healing. We developed a microcomposite substrate (μCS) platform with a microfabricated base and collagen hydrogel top to generate physiological linear stiffness gradients without any variation in chemical or transport properties. This platform is compatible with both 2D and 3D cell culturing and can be assembled with common supplies found in most biology labs. Ligament fibroblasts (LFs) and mesenchymal stem cells (MSCs) both respond to the mechanical gradient with directed migration. Interestingly, LFs exhibit higher mechanosensitivity compared with MSCs. Polarized nonmuscle myosin IIB distribution was also found on the μCS gradient, confirming previous reports. This robust system provides an easily accessible platform to study cell mechanosensing and a more physiological microenvironment for cell studies. Copyright © 2014 Elsevier Ltd. All rights reserved.

Phenotypic research on senile osteoporosis caused by SIRT6 deficiency

PubMed Central

Zhang, De-Mao; Cui, Di-Xin; Xu, Ruo-Shi; Zhou, Ya-Chuan; Zheng, Li-Wei; Liu, Peng; Zhou, Xue-Dong

2016-01-01

Osteoporosis is a serious public bone metabolic disease. However, the mechanisms underlying bone loss combined with ageing, which is known as senile osteoporosis, remains unknown. Here we show the detailed phenotype of this disease caused by SIRT6 knock out (KO) in mice. To the best of our knowledge, this is the first study to reveal that SIRT6 is expressed in both bone marrow stroma cells and bone-related cells in both mouse and human models, which suggests that SIRT6 is an important regulator in bone metabolism. SIRT6-KO mice exhibit a significant decrease in body weight and remarkable dwarfism. The skeleton of the SIRT6-KO mouse is deficient in cartilage and mineralized bone tissue. Moreover, the osteocalcin concentration in blood is lower, which suggests that bone mass is markedly lost. Besides, the tartrate-resistant acid phosphatase 5b (TRAP5b) concentration is much higher, which suggests that bone resorption is overactive. Both trabecular and cortical bones exhibit severe osteopenia, and the bone mineral density is decreased. Moreover, double-labelling analysis shows that bone formation is much slower. To determine whether SIRT6 directly regulates bone metabolism, we cultured primary bone marrow stromal cells for osteogenesis and osteoclastogenesis separately to avoid indirect interference in vivo responses such as inflammation. Taken together, these results show that SIRT6 can directly regulate osteoblast proliferation and differentiation, resulting in attenuation in mineralization. Furthermore, SIRT6 can directly regulate osteoclast differentiation and results in a higher number of small osteoclasts, which may be related to overactive bone resorption. PMID:27357320

Building predictive in vitro pulmonary toxicity assays using high-throughput imaging and artificial intelligence.

PubMed

Lee, Jia-Ying Joey; Miller, James Alastair; Basu, Sreetama; Kee, Ting-Zhen Vanessa; Loo, Lit-Hsin

2018-06-01

Human lungs are susceptible to the toxicity induced by soluble xenobiotics. However, the direct cellular effects of many pulmonotoxic chemicals are not always clear, and thus, a general in vitro assay for testing pulmonotoxicity applicable to a wide variety of chemicals is not currently available. Here, we report a study that uses high-throughput imaging and artificial intelligence to build an in vitro pulmonotoxicity assay by automatically comparing and selecting human lung-cell lines and their associated quantitative phenotypic features most predictive of in vivo pulmonotoxicity. This approach is called "High-throughput In vitro Phenotypic Profiling for Toxicity Prediction" (HIPPTox). We found that the resulting assay based on two phenotypic features of a human bronchial epithelial cell line, BEAS-2B, can accurately classify 33 reference chemicals with human pulmonotoxicity information (88.8% balance accuracy, 84.6% sensitivity, and 93.0% specificity). In comparison, the predictivity of a standard cell-viability assay on the same set of chemicals is much lower (77.1% balanced accuracy, 84.6% sensitivity, and 69.5% specificity). We also used the assay to evaluate 17 additional test chemicals with unknown/unclear human pulmonotoxicity, and experimentally confirmed that many of the pulmonotoxic reference and predicted-positive test chemicals induce DNA strand breaks and/or activation of the DNA-damage response (DDR) pathway. Therefore, HIPPTox helps us to uncover these common modes-of-action of pulmonotoxic chemicals. HIPPTox may also be applied to other cell types or models, and accelerate the development of predictive in vitro assays for other cell-type- or organ-specific toxicities.

Cellular Dichotomy Between Anchorage-Independent Growth Responses to bFGF and TA Reflects Molecular Switch in Commitment to Carcinogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Waters, Katrina M.; Tan, Ruimin; Opresko, Lee K.

2009-11-01

We have investigated gene expression patterns underlying reversible and irreversible anchorage-independent growth (AIG) phenotypes to identify more sensitive markers of cell transformation for studies directed at interrogating carcinogenesis responses. In JB6 mouse epidermal cells, basic fibroblast growth factor (bFGF) induces an unusually efficient and reversible AIG response, relative to 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced AIG which is irreversible. The reversible and irreversible AIG phenotypes are characterized by largely non-overlapping global gene expression profiles. However, a subset of differentially expressed genes were identified as common to reversible and irreversible AIG phenotypes, including genes regulated in a reciprocal fashion. Hepatic leukemia factor (HLF) andmore » D-site albumin promoter-binding protein (DBP) were increased in both bFGF and TPA soft agar colonies and selected for functional validation. Ectopic expression of human HLF and DBP in JB6 cells resulted in a marked increase in TPA- and bFGF-regulated AIG responses. HLF and DBP expression were increased in soft agar colonies arising from JB6 cells exposed to gamma radiation and in a human basal cell carcinoma tumor tissue, relative to paired non-tumor tissue. Subsequent biological network analysis suggests that many of the differentially expressed genes that are common to bFGF- and TPA-dependent AIG are regulated by c-Myc, SP-1 and HNF-4 transcription factors. Collectively, we have identified a potential molecular switch that mediates the transition from reversible to irreversible AIG.« less

Cryopreservation of human vascular umbilical cord cells under good manufacturing practice conditions for future cell banks.

PubMed

Polchow, Bianca; Kebbel, Kati; Schmiedeknecht, Gerno; Reichardt, Anne; Henrich, Wolfgang; Hetzer, Roland; Lueders, Cora

2012-05-16

In vitro fabricated tissue engineered vascular constructs could provide an alternative to conventional substitutes. A crucial factor for tissue engineering of vascular constructs is an appropriate cell source. Vascular cells from the human umbilical cord can be directly isolated and cryopreserved until needed. Currently no cell bank for human vascular cells is available. Therefore, the establishment of a future human vascular cell bank conforming to good manufacturing practice (GMP) conditions is desirable for therapeutic applications such as tissue engineered cardiovascular constructs. A fundamental step was the adaption of conventional research and development starting materials to GMP compliant starting materials. Human umbilical cord artery derived cells (HUCAC) and human umbilical vein endothelial cells (HUVEC) were isolated, cultivated, cryopreserved (short- and long-term) directly after primary culture and recultivated subsequently. Cell viability, expression of cellular markers and proliferation potential of fresh and cryopreserved cells were studied using trypan blue staining, flow cytometry analysis, immunofluorescence staining and proliferation assays. Statistical analyses were performed using Student's t-test. Sufficient numbers of isolated cells with acceptable viabilities and homogenous expression of cellular markers confirmed that the isolation procedure was successful using GMP compliant starting materials. The influence of cryopreservation was marginal, because cryopreserved cells mostly maintain phenotypic and functional characteristics similar to those of fresh cells. Phenotypic studies revealed that fresh cultivated and cryopreserved HUCAC were positive for alpha smooth muscle actin, CD90, CD105, CD73, CD29, CD44, CD166 and negative for smoothelin. HUVEC expressed CD31, CD146, CD105 and CD144 but not alpha smooth muscle actin. Functional analysis demonstrated acceptable viability and sufficient proliferation properties of cryopreserved HUCAC and HUVEC. Adaptation of cell isolation, cultivation and cryopreservation to GMP compliant starting materials was successful. Cryopreservation did not influence cell properties with lasting impact, confirming that the application of vascular cells from the human umbilical cord is feasible for cell banking. A specific cellular marker expression profile was established for HUCAC and HUVEC using flow cytometry analysis, applicable as a GMP compliant quality control. Use of these cells for the future fabrication of advanced therapy medicinal products GMP conditions are required by the regulatory authority.

A Novel Strategy to Increase the Proliferative Potential of Adult Human β-Cells While Maintaining Their Differentiated Phenotype

PubMed Central

Aly, Haytham; Rohatgi, Nidhi; Marshall, Connie A.; Grossenheider, Tiffani C.; Miyoshi, Hiroyuki; Stappenbeck, Thaddeus S.; Matkovich, Scot J.; McDaniel, Michael L.

2013-01-01

Our previous studies demonstrated that Wnt/GSK-3/β-catenin and mTOR signaling are necessary to stimulate proliferative processes in adult human β-cells. Direct inhibition of GSK-3, that engages Wnt signaling downstream of the Wnt receptor, increases β-catenin nuclear translocation and β-cell proliferation but results in lower insulin content. Our current goal was to engage canonical and non-canonical Wnt signaling at the receptor level to significantly increase human β-cell proliferation while maintaining a β-cell phenotype in intact islets. We adopted a system that utilized conditioned medium from L cells that expressed Wnt3a, R-spondin-3 and Noggin (L-WRN conditioned medium). In addition we used a ROCK inhibitor (Y-27632) and SB-431542 (that results in RhoA inhibition) in these cultures. Treatment of intact human islets with L-WRN conditioned medium plus inhibitors significantly increased DNA synthesis ∼6 fold in a rapamycin-sensitive manner. Moreover, this treatment strikingly increased human β-cell proliferation ∼20 fold above glucose alone. Only the combination of L-WRN conditioned medium with RhoA/ROCK inhibitors resulted in substantial proliferation. Transcriptome-wide gene expression profiling demonstrated that L-WRN medium provoked robust changes in several signaling families, including enhanced β-catenin-mediated and β-cell-specific gene expression. This treatment also increased expression of Nr4a2 and Irs2 and resulted in phosphorylation of Akt. Importantly, glucose-stimulated insulin secretion and content were not downregulated by L-WRN medium treatment. Our data demonstrate that engaging Wnt signaling at the receptor level by this method leads to necessary crosstalk between multiple signaling pathways including activation of Akt, mTOR, Wnt/β-catenin, PKA/CREB, and inhibition of RhoA/ROCK that substantially increase human β-cell proliferation while maintaining the β-cell phenotype. PMID:23776620

A novel strategy to increase the proliferative potential of adult human β-cells while maintaining their differentiated phenotype.

PubMed

Aly, Haytham; Rohatgi, Nidhi; Marshall, Connie A; Grossenheider, Tiffani C; Miyoshi, Hiroyuki; Stappenbeck, Thaddeus S; Matkovich, Scot J; McDaniel, Michael L

2013-01-01

Our previous studies demonstrated that Wnt/GSK-3/β-catenin and mTOR signaling are necessary to stimulate proliferative processes in adult human β-cells. Direct inhibition of GSK-3, that engages Wnt signaling downstream of the Wnt receptor, increases β-catenin nuclear translocation and β-cell proliferation but results in lower insulin content. Our current goal was to engage canonical and non-canonical Wnt signaling at the receptor level to significantly increase human β-cell proliferation while maintaining a β-cell phenotype in intact islets. We adopted a system that utilized conditioned medium from L cells that expressed Wnt3a, R-spondin-3 and Noggin (L-WRN conditioned medium). In addition we used a ROCK inhibitor (Y-27632) and SB-431542 (that results in RhoA inhibition) in these cultures. Treatment of intact human islets with L-WRN conditioned medium plus inhibitors significantly increased DNA synthesis ∼6 fold in a rapamycin-sensitive manner. Moreover, this treatment strikingly increased human β-cell proliferation ∼20 fold above glucose alone. Only the combination of L-WRN conditioned medium with RhoA/ROCK inhibitors resulted in substantial proliferation. Transcriptome-wide gene expression profiling demonstrated that L-WRN medium provoked robust changes in several signaling families, including enhanced β-catenin-mediated and β-cell-specific gene expression. This treatment also increased expression of Nr4a2 and Irs2 and resulted in phosphorylation of Akt. Importantly, glucose-stimulated insulin secretion and content were not downregulated by L-WRN medium treatment. Our data demonstrate that engaging Wnt signaling at the receptor level by this method leads to necessary crosstalk between multiple signaling pathways including activation of Akt, mTOR, Wnt/β-catenin, PKA/CREB, and inhibition of RhoA/ROCK that substantially increase human β-cell proliferation while maintaining the β-cell phenotype.

PRDM14 directly interacts with heat shock proteins HSP90α and glucose-regulated protein 78.

PubMed

Moriya, Chiharu; Taniguchi, Hiroaki; Nagatoishi, Satoru; Igarashi, Hisayoshi; Tsumoto, Kouhei; Imai, Kohzoh

2018-02-01

PRDM14 is overexpressed in various cancers and can regulate cancer phenotype under certain conditions. Inhibiting PRDM14 expression in breast and pancreatic cancers has been reported to reduce cancer stem-like phenotypes, which are associated with aggressive tumor properties. Therefore, PRDM14 is considered a promising target for cancer therapy. To develop a pharmaceutical treatment, the mechanism and interacting partners of PRDM14 need to be clarified. Here, we identified the proteins interacting with PRDM14 in triple-negative breast cancer (TNBC) cells, which do not express the three most common types of receptor (estrogen receptors, progesterone receptors, and HER2). We obtained 13 candidates that were pulled down with PRDM14 in TNBC HCC1937 cells and identified them by mass spectrometry. Two candidates-glucose-regulated protein 78 (GRP78) and heat shock protein 90-α (HSP90α)-were confirmed in immunoprecipitation assay in two TNBC cell lines (HCC1937 and MDA-MB231). Surface plasmon resonance analysis using GST-PRDM14 showed that these two proteins directly interacted with PRDM14 and that the interactions required the C-terminal region of PRDM14, which includes zinc finger motifs. We also confirmed the interactions in living cells by NanoLuc luciferase-based bioluminescence resonance energy transfer (NanoBRET) assay. Moreover, HSP90 inhibitors (17DMAG and HSP990) significantly decreased breast cancer stem-like CD24 -  CD44 + and side population (SP) cells in HCC1937 cells, but not in PRDM14 knockdown HCC1937 cells. The combination of the GRP78 inhibitor HA15 and PRDM14 knockdown significantly decreased cell proliferation and SP cell number in HCC1937 cells. These results suggest that HSP90α and GRP78 interact with PRDM14 and participate in cancer regulation. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Analysis of cell cycle-related proteins in gastric intramucosal differentiated-type cancers based on mucin phenotypes: a novel hypothesis of early gastric carcinogenesis based on mucin phenotype

PubMed Central

2010-01-01

Background Abnormalities of cell cycle regulators are common features in human cancers, and several of these factors are associated with the early development of gastric cancers. However, recent studies have shown that gastric cancer tumorigenesis was characterized by mucin expression. Thus, expression patterns of cell cycle-related proteins were investigated in the early phase of differentiated-type gastric cancers to ascertain any mechanistic relationships with mucin phenotypes. Methods Immunostaining for Cyclins D1, A, E, and p21, p27, p53 and β-catenin was used to examine impairments of the cell cycle in 190 gastric intramucosal differentiated-type cancers. Mucin phenotypes were determined by the expressions of MUC5AC, MUC6, MUC2 and CD10. A Ki-67 positive rate (PR) was also examined. Results Overexpressions of p53, cyclin D1 and cyclin A were significantly more frequent in a gastric phenotype than an intestinal phenotype. Cyclin A was overexpressed in a mixed phenotype compared with an intestinal phenotype, while p27 overexpression was more frequent in an intestinal phenotype than in a mixed phenotype. Reduction of p21 was a common feature of the gastric intramucosal differentiated-type cancers examined. Conclusions Our results suggest that the levels of some cell cycle regulators appear to be associated with mucin phenotypes of early gastric differentiated-type cancers. PMID:20525401

Calcium as a cardiovascular toxin in CKD-MBD.

PubMed

Moe, Sharon M

2017-07-01

Disordered calcium balance and homeostasis are common in patients with chronic kidney disease. Such alterations are commonly associated with abnormal bone remodeling, directly and indirectly. Similarly, positive calcium balance may also be a factor in the pathogenesis of extra skeletal soft tissue and arterial calcification. Calcium may directly affect cardiac structure and function through direct effects to alter cell signaling due to abnormal intracellular calcium homeostasis 2) extra-skeletal deposition of calcium and phosphate in the myocardium and small cardiac arterioles, 3) inducing cardiomyocyte hypertrophy through calcium and hormone activation of NFAT signaling mechanisms, and 4) increased aorta calcification resulting in chronic increased afterload leading to hypertrophy. Similarly, calcium may alter vascular smooth muscle cell function and affect cell signaling which may predispose to a proliferative phenotype important in arteriosclerosis and arterial calcification. Thus, disorders of calcium balance and homeostasis due to CKD-MBD may play a role in the high cardiovascular burden observed in patients with CKD. Published by Elsevier Inc.

In Vitro Assays for Mouse Müller Cell Phenotyping Through microRNA Profiling in the Damaged Retina.

PubMed

Reyes-Aguirre, Luis I; Quintero, Heberto; Estrada-Leyva, Brenda; Lamas, Mónica

2018-01-01

microRNA profiling has identified cell-specific expression patterns that could represent molecular signatures triggering the acquisition of a specific phenotype; in other words, of cellular identity and its associated function. Several groups have hypothesized that retinal cell phenotyping could be achieved through the determination of the global pattern of miRNA expression across specific cell types in the adult retina. This is especially relevant for Müller glia in the context of retinal damage, as these cells undergo dramatic changes of gene expression in response to injury, that render them susceptible to acquire a progenitor-like phenotype and be a source of new neurons.We describe a method that combines an experimental protocol for excitotoxic-induced retinal damage through N-methyl-D-aspartate subretinal injection with magnetic-activated cell sorting (MACS) of Müller cells and RNA isolation for microRNA profiling. Comparison of microRNA patterns of expression should allow Müller cell phenotyping under different experimental conditions.

Increased alternative lengthening of telomere phenotypes of telomerase-negative immortal cells upon trichostatin--a treatment.

PubMed

Jung, A Ra; Yoo, Jeong Eun; Shim, Yhong-Hee; Choi, Ye-Na; Jeung, Hei-Cheul; Chung, Hyun Cheol; Rha, Sun Young; Oh, Bong-Kyeong

2013-03-01

Human immortal cells maintain their telomeres either by telomerase or by alternative lengthening of telomeres (ALT) that is based on homologous telomeric recombination. Previous studies showed that the ALT mechanism is activated in non-ALT cells when heterochromatic features are reduced. In this study, we examined the ALT phenotypes of ALT cells after treatment with trichostatin-A (TSA), which is an inhibitor of histone deacetylases and causes global chromatin decondensation. The ALT cells remained telomerase-negative after TSA treatment. ALT-associated promyelocytic leukemia (PML) nuclear bodies and telomere sister chromatid exchanges, typical ALT phenotypes, markedly increased in the TSA-treated cells, while the telomere length remained unchanged. In addition, telomerase expression in the ALT cells suppressed TSA-mediated ALT phenotype enhancement. Our results show that certain ALT phenotypes become more pronounced when chromatin is decondensed, and also suggest that the ALT mechanism may compete with telomerase for telomere maintenance in cells that lack heterochromatin.

Directing bone marrow-derived stromal cell function with mechanics.

PubMed

Potier, E; Noailly, J; Ito, K

2010-03-22

Because bone marrow-derived stromal cells (BMSCs) are able to generate many cell types, they are envisioned as source of regenerative cells to repair numerous tissues, including bone, cartilage, and ligaments. Success of BMSC-based therapies, however, relies on a number of methodological improvements, among which better understanding and control of the BMSC differentiation pathways. Since many years, the biochemical environment is known to govern BMSC differentiation, but more recent evidences show that the biomechanical environment is also directing cell functions. Using in vitro systems that aim to reproduce selected components of the in vivo mechanical environment, it was demonstrated that mechanical loadings can affect BMSC proliferation and improve the osteogenic, chondrogenic, or myogenic phenotype of BMSCs. These effects, however, seem to be modulated by parameters other than mechanics, such as substrate nature or soluble biochemical environment. This paper reviews and discusses recent experimental data showing that despite some knowledge limitation, mechanical stimulation already constitutes an additional and efficient tool to drive BMSC differentiation. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Dominant-negative mutants of platelet-derived growth factor revert the transformed phenotype of human astrocytoma cells.

PubMed Central

Shamah, S M; Stiles, C D; Guha, A

1993-01-01

Malignant astrocytoma is the most common primary human brain tumor. Most astrocytomas express a combination of platelet-derived growth factor (PDGF) and PDGF receptor which could close an autocrine loop. It is not known whether these autocrine loops contribute to the transformed phenotype of astrocytoma cells or are incidental to that phenotype. Here we show that dominant-negative mutants of the PDGF ligand break the autocrine loop and revert the phenotype of BALB/c 3T3 cells transformed by the PDGF-A or PDGF-B (c-sis) gene. Then, we show that these mutants are selective in that they do not alter the phenotype of 3T3 cells transformed by an activated Ha-ras or v-src gene or by simian virus 40. Finally, we show that these mutants revert the transformed phenotype of two independent human astrocytoma cell lines. They have no effect on the growth of human medulloblastoma, bladder carcinoma, or colon carcinoma cell lines. These observations are consistent with the view that PDGF autocrine loops contribute to the transformed phenotype of at least some human astrocytomas. Images PMID:8246942

Oncogenic Viruses and Tumor Glucose Metabolism: Like Kids in a Candy Store

PubMed Central

Noch, Evan; Khalili, Kamel

2011-01-01

Oncogenic viruses represent a significant public health burden in light of the multitude of malignancies resulting from chronic or spontaneous viral infection and transformation. Though many of the molecular signaling pathways underlying virus-mediated cellular transformation are known, the impact of these viruses on metabolic signaling and phenotype within proliferating tumor cells is less well understood. Whether the interaction of oncogenic viruses with metabolic signaling pathways involves enhanced glucose uptake and glycolysis, both hallmark features of transformed cells, or dysregulation of molecular pathways regulating oxidative stress, viruses are adept at facilitating tumor expansion. Through their effects on cell proliferation pathways, such as the PI3K and MAPK pathways, the cell cycle regulatory proteins, p53 and ATM, and the cell stress response proteins, HIF-1α and AMPK, viruses exert control over critical metabolic signaling cascades. Additionally, oncogenic viruses modulate the tumor metabolomic profile through direct and indirect interaction with glucose transporters, such as GLUT1, and specific glycolytic enzymes, including pyruvate kinase, glucose 6-phosphate dehydrogenase, and hexokinase. Through these pathways, oncogenic viruses alter the phenotypic characteristics of transformed cells and their methods of energy utilization, and it may be possible to develop novel anti-glycolytic therapies to target these dysregulated pathways in virus-derived malignancies. PMID:22234809

The Effects of Antigen-Specific IgG1 Antibody for the Pulmonary-Hypertension-Phenotype and B Cells for Inflammation in Mice Exposed to Antigen and Fine Particles from Air Pollution

PubMed Central

Park, Sung-Hyun; Chen, Wen-Chi; Durmus, Nedim; Bleck, Bertram; Reibman, Joan; Riemekasten, Gabriela; Grunig, Gabriele

2015-01-01

Air pollution is known to exacerbate chronic inflammatory conditions of the lungs including pulmonary hypertension, cardiovascular diseases and autoimmune diseases. Directly pathogenic antibodies bind pro-inflammatory cell receptors and cause or exacerbate inflammation. In contrast, anti-inflammatory antibody isotypes (e.g. mouse immunoglobulin G1, IgG1) bind inhibitory cell receptors and can inhibit inflammation. Our previous studies showed that co-exposure to antigen and urban ambient particulate matter (PM2.5) induced severe pulmonary arterial thickening and increased right ventricular systolic pressures in mice via T-cell produced cytokines, Interleukin (IL)-13 and IL-17A. The aim of the current study was to understand how B cell and antibody responses integrate into this T cell cytokine network for the pulmonary hypertension phenotype. Special focus was on antigen-specific IgG1 that is the predominant antibody in the experimental response to antigen and urban ambient PM2.5. Wild type and B cell-deficient mice were primed with antigen and then challenged with antigen and urban particulate matter and injected with antibodies as appropriate. Our data surprisingly showed that B cells were necessary for the development of increased right ventricular pressures and molecular changes in the right heart in response to sensitization and intranasal challenge with antigen and PM2.5. Further, our studies showed that both, the increase in right ventricular systolic pressure and right ventricular molecular changes were restored by reconstituting the B cell KO mice with antigen specific IgG1. In addition, our studies identified a critical, non-redundant role of B cells for the IL-17A-directed inflammation in response to exposure with antigen and PM2.5, which was not corrected with antigen-specific IgG1. In contrast, IL-13-directed inflammatory markers, as well as severe pulmonary arterial remodeling induced by challenge with antigen and PM2.5 were similar in B cell-deficient and wild type mice. Our studies have identified B cells and antigen specific IgG1 as potential therapeutic targets for pulmonary hypertension associated with immune dysfunction and environmental exposures. PMID:26079807

Evidence for complete epistasis of null mutations in murine Fanconi anemia genes Fanca and Fancg.

PubMed

van de Vrugt, Henri J; Koomen, Mireille; Bakker, Sietske; Berns, Mariska A D; Cheng, Ngan Ching; van der Valk, Martin A; de Vries, Yne; Rooimans, Martin A; Oostra, Anneke B; Hoatlin, Maureen E; Te Riele, Hein; Joenje, Hans; Arwert, Fré

2011-12-10

Fanconi anemia (FA) is a heritable disease characterized by bone marrow failure, congenital abnormalities, and cancer predisposition. The 15 identified FA genes operate in a molecular pathway to preserve genomic integrity. Within this pathway the FA core complex operates as an ubiquitin ligase that activates the complex of FANCD2 and FANCI to coordinate DNA repair. The FA core complex is formed by at least 12 proteins. However, only the FANCL subunit displays ubiquitin ligase activity. FANCA and FANCG are members of the FA core complex for which no other functions have been described than to participate in protein interactions. In this study we generated mice with combined null alleles for Fanca and Fancg to identify extended functions for these genes by characterizing the double mutant mice and cells. Double mutant a(-/-)/g(-/-) mice were born at near Mendelian frequencies without apparent developmental abnormalities. Histological analysis of a(-/-)/g(-/-) mice revealed a Leydig cell hyperplasia and frequent vacuolization of Sertoli cells in testes, while ovaries were depleted from developing follicles and displayed an interstitial cell hyperplasia. These gonadal aberrations were associated with a compromised fertility of a(-/-)/g(-/-) males and females. During the first year of life a(-/-)/g(-/-) did not develop malignancies or bone marrow failure. At the cellular level a(-/-)/g(-/-), Fanca(-/-), and Fancg(-/-) cells proved equally compromised in DNA crosslink and homology-directed repair. Overall the phenotype of a(-/-)/g(-/-) double knockout mice and cells appeared highly similar to the phenotype of Fanca or Fancg single knockouts. The lack of an augmented phenotype suggest that null mutations in Fanca or Fancg are fully epistatic, making additional important functions outside of the FA core complex highly unlikely. 2011 Elsevier B.V. All rights reserved.

Pericellular Versican Regulates the Fibroblast-Myofibroblast Transition

PubMed Central

Hattori, Noriko; Carrino, David A.; Lauer, Mark E.; Vasanji, Amit; Wylie, James D.; Nelson, Courtney M.; Apte, Suneel S.

2011-01-01

The cell and its glycosaminoglycan-rich pericellular matrix (PCM) comprise a functional unit. Because modification of PCM influences cell behavior, we investigated molecular mechanisms that regulate PCM volume and composition. In fibroblasts and other cells, aggregates of hyaluronan and versican are found in the PCM. Dermal fibroblasts from Adamts5−/− mice, which lack a versican-degrading protease, ADAMTS5, had reduced versican proteolysis, increased PCM, altered cell shape, enhanced α-smooth muscle actin (SMA) expression and increased contractility within three-dimensional collagen gels. The myofibroblast-like phenotype was associated with activation of TGFβ signaling. We tested the hypothesis that fibroblast-myofibroblast transition in Adamts5−/− cells resulted from versican accumulation in PCM. First, we noted that versican overexpression in human dermal fibroblasts led to increased SMA expression, enhanced contractility, and increased Smad2 phosphorylation. In contrast, dermal fibroblasts from Vcan haploinsufficient (Vcanhdf/+) mice had reduced contractility relative to wild type fibroblasts. Using a genetic approach to directly test if myofibroblast transition in Adamts5−/− cells resulted from increased PCM versican content, we generated Adamts5−/−;Vcanhdf/+ mice and isolated their dermal fibroblasts for comparison with dermal fibroblasts from Adamts5−/− mice. In Adamts5−/− fibroblasts, Vcan haploinsufficiency or exogenous ADAMTS5 restored normal fibroblast contractility. These findings demonstrate that altering PCM versican content through proteolytic activity of ADAMTS5 profoundly influenced the dermal fibroblast phenotype and may regulate a phenotypic continuum between the fibroblast and its alter ego, the myofibroblast. We propose that a physiological function of ADAMTS5 in dermal fibroblasts is to maintain optimal versican content and PCM volume by continually trimming versican in hyaluronan-versican aggregates. PMID:21828051

TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts.

PubMed

Bernstein, Diana L; Le Lay, John E; Ruano, Elena G; Kaestner, Klaus H

2015-05-01

Current strategies to alter disease-associated epigenetic modifications target ubiquitously expressed epigenetic regulators. This approach does not allow specific genes to be controlled in specific cell types; therefore, tools to selectively target epigenetic modifications in the desired cell type and strategies to more efficiently correct aberrant gene expression in disease are needed. Here, we have developed a method for directing DNA methylation to specific gene loci by conjugating catalytic domains of DNA methyltransferases (DNMTs) to engineered transcription activator-like effectors (TALEs). We demonstrated that these TALE-DNMTs direct DNA methylation specifically to the targeted gene locus in human cells. Further, we determined that minimizing direct nucleotide sequence repeats within the TALE moiety permits efficient lentivirus transduction, allowing easy targeting of primary cell types. Finally, we demonstrated that directed DNA methylation with a TALE-DNMT targeting the CDKN2A locus, which encodes the cyclin-dependent kinase inhibitor p16, decreased CDKN2A expression and increased replication of primary human fibroblasts, as intended. Moreover, overexpression of p16 in these cells reversed the proliferative phenotype, demonstrating the specificity of our epigenetic targeting. Together, our results demonstrate that TALE-DNMTs can selectively target specific genes and suggest that this strategy has potential application for the development of locus-specific epigenetic therapeutics.

TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts

PubMed Central

Bernstein, Diana L.; Le Lay, John E.; Ruano, Elena G.; Kaestner, Klaus H.

2015-01-01

Current strategies to alter disease-associated epigenetic modifications target ubiquitously expressed epigenetic regulators. This approach does not allow specific genes to be controlled in specific cell types; therefore, tools to selectively target epigenetic modifications in the desired cell type and strategies to more efficiently correct aberrant gene expression in disease are needed. Here, we have developed a method for directing DNA methylation to specific gene loci by conjugating catalytic domains of DNA methyltransferases (DNMTs) to engineered transcription activator–like effectors (TALEs). We demonstrated that these TALE-DNMTs direct DNA methylation specifically to the targeted gene locus in human cells. Further, we determined that minimizing direct nucleotide sequence repeats within the TALE moiety permits efficient lentivirus transduction, allowing easy targeting of primary cell types. Finally, we demonstrated that directed DNA methylation with a TALE-DNMT targeting the CDKN2A locus, which encodes the cyclin-dependent kinase inhibitor p16, decreased CDKN2A expression and increased replication of primary human fibroblasts, as intended. Moreover, overexpression of p16 in these cells reversed the proliferative phenotype, demonstrating the specificity of our epigenetic targeting. Together, our results demonstrate that TALE-DNMTs can selectively target specific genes and suggest that this strategy has potential application for the development of locus-specific epigenetic therapeutics. PMID:25866970

Concordant but Varied Phenotypes among Duchenne Muscular Dystrophy Patient-Specific Myoblasts Derived using a Human iPSC-Based Model.

PubMed

Choi, In Young; Lim, HoTae; Estrellas, Kenneth; Mula, Jyothi; Cohen, Tatiana V; Zhang, Yuanfan; Donnelly, Christopher J; Richard, Jean-Philippe; Kim, Yong Jun; Kim, Hyesoo; Kazuki, Yasuhiro; Oshimura, Mitsuo; Li, Hongmei Lisa; Hotta, Akitsu; Rothstein, Jeffrey; Maragakis, Nicholas; Wagner, Kathryn R; Lee, Gabsang

2016-06-07

Duchenne muscular dystrophy (DMD) remains an intractable genetic disease. Althogh there are several animal models of DMD, there is no human cell model that carries patient-specific DYSTROPHIN mutations. Here, we present a human DMD model using human induced pluripotent stem cells (hiPSCs). Our model reveals concordant disease-related phenotypes with patient-dependent variation, which are partially reversed by genetic and pharmacological approaches. Our "chemical-compound-based" strategy successfully directs hiPSCs into expandable myoblasts, which exhibit a myogenic transcriptional program, forming striated contractile myofibers and participating in muscle regeneration in vivo. DMD-hiPSC-derived myoblasts show disease-related phenotypes with patient-to-patient variability, including aberrant expression of inflammation or immune-response genes and collagens, increased BMP/TGFβ signaling, and reduced fusion competence. Furthermore, by genetic correction and pharmacological "dual-SMAD" inhibition, the DMD-hiPSC-derived myoblasts and genetically corrected isogenic myoblasts form "rescued" multi-nucleated myotubes. In conclusion, our findings demonstrate the feasibility of establishing a human "DMD-in-a-dish" model using hiPSC-based disease modeling. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

The chromokinesin Kid is required for maintenance of proper metaphase spindle size.

PubMed

Tokai-Nishizumi, Noriko; Ohsugi, Miho; Suzuki, Emiko; Yamamoto, Tadashi

2005-11-01

The human chromokinesin Kid/kinesin-10, a plus end-directed microtubule (MT)-based motor with both microtubule- and DNA-binding domains, is required for proper chromosome alignment at the metaphase plate. Here, we performed RNA interference experiments to deplete endogenous Kid from HeLa cells and confirmed defects in metaphase chromosome arm alignment in Kid-depleted cells. In addition, we noted a shortening of the spindle length, resulting in a pole-to-pole distance only 80% of wild type. The spindle microtubule-bundles with which Kid normally colocalize became less robust. Rescue of the two Kid deficiency phenotypes-imprecise chromosome alignment at metaphase and shortened spindles- exhibited distinct requirements. Mutants lacking either the DNA-binding domain or the MT motor ATPase failed to rescue the former defect, whereas rescue of the shortened spindle phenotype required neither activity. Kid also exhibits microtubule bundling activity in vitro, and rescue of the shortened spindle phenotype and the bundling activity displayed similar domain requirements, except that rescue required a coiled-coil domain not needed for bundling. These results suggest that distinct from its role in chromosome movement, Kid contributes to spindle morphogenesis by mediating spindle microtubules stabilization.

Exosomes derived from mesenchymal non-small cell lung cancer cells promote chemoresistance.

PubMed

Lobb, Richard J; van Amerongen, Rosa; Wiegmans, Adrian; Ham, Sunyoung; Larsen, Jill E; Möller, Andreas

2017-08-01

Non-small cell lung cancer (NSCLC) is the most common lung cancer type and the most common cause of mortality in lung cancer patients. NSCLC is often associated with resistance to chemotherapeutics and together with rapid metastatic spread, results in limited treatment options and poor patient survival. NSCLCs are heterogeneous, and consist of epithelial and mesenchymal NSCLC cells. Mesenchymal NSCLC cells are thought to be responsible for the chemoresistance phenotype, but if and how this phenotype can be transferred to other NSCLC cells is currently not known. We hypothesised that small extracellular vesicles, exosomes, secreted by mesenchymal NSCLC cells could potentially transfer the chemoresistance phenotype to surrounding epithelial NSCLC cells. To explore this possibility, we used a unique human bronchial epithelial cell (HBEC) model in which the parental cells were transformed from an epithelial to mesenchymal phenotype by introducing oncogenic alterations common in NSCLC. We found that exosomes derived from the oncogenically transformed, mesenchymal HBECs could transfer chemoresistance to the parental, epithelial HBECs and increase ZEB1 mRNA, a master EMT transcription factor, in the recipient cells. Additionally, we demonstrate that exosomes from mesenchymal, but not epithelial HBECs contain the ZEB1 mRNA, thereby providing a potential mechanism for the induction of a mesenchymal phenotype in recipient cells. Together, this work demonstrates for the first time that exosomes derived from mesenchymal, oncogenically transformed lung cells can transfer chemoresistance and mesenchymal phenotypes to recipient cells, likely via the transfer of ZEB1 mRNA in exosomes. © 2017 UICC.

Spontaneous In Vivo Chondrogenesis of Bone Marrow-Derived Mesenchymal Progenitor Cells by Blocking Vascular Endothelial Growth Factor Signaling.

PubMed

Marsano, Anna; Medeiros da Cunha, Carolina M; Ghanaati, Shahram; Gueven, Sinan; Centola, Matteo; Tsaryk, Roman; Barbeck, Mike; Stuedle, Chiara; Barbero, Andrea; Helmrich, Uta; Schaeren, Stefan; Kirkpatrick, James C; Banfi, Andrea; Martin, Ivan

2016-12-01

: Chondrogenic differentiation of bone marrow-derived mesenchymal stromal/stem cells (MSCs) can be induced by presenting morphogenetic factors or soluble signals but typically suffers from limited efficiency, reproducibility across primary batches, and maintenance of phenotypic stability. Considering the avascular and hypoxic milieu of articular cartilage, we hypothesized that sole inhibition of angiogenesis can provide physiological cues to direct in vivo differentiation of uncommitted MSCs to stable cartilage formation. Human MSCs were retrovirally transduced to express a decoy soluble vascular endothelial growth factor (VEGF) receptor-2 (sFlk1), which efficiently sequesters endogenous VEGF in vivo, seeded on collagen sponges and immediately implanted ectopically in nude mice. Although naïve cells formed vascularized fibrous tissue, sFlk1-MSCs abolished vascular ingrowth into engineered constructs, which efficiently and reproducibly developed into hyaline cartilage. The generated cartilage was phenotypically stable and showed no sign of hypertrophic evolution up to 12 weeks. In vitro analyses indicated that spontaneous chondrogenic differentiation by blockade of angiogenesis was related to the generation of a hypoxic environment, in turn activating the transforming growth factor-β pathway. These findings suggest that VEGF blockade is a robust strategy to enhance cartilage repair by endogenous or grafted mesenchymal progenitors. This article outlines the general paradigm of controlling the fate of implanted stem/progenitor cells by engineering their ability to establish specific microenvironmental conditions rather than directly providing individual morphogenic cues. Chondrogenic differentiation of mesenchymal stromal/stem cells (MSCs) is typically targeted by morphogen delivery, which is often associated with limited efficiency, stability, and robustness. This article proposes a strategy to engineer MSCs with the capacity to establish specific microenvironmental conditions, supporting their own targeted differentiation program. Sole blockade of angiogenesis mediated by transduction for sFlk-1, without delivery of additional morphogens, is sufficient for inducing MSC chondrogenic differentiation. The findings represent a relevant step forward in the field because the method allowed reducing interdonor variability in MSC differentiation efficiency and, importantly, onset of a stable, nonhypertrophic chondrocyte phenotype. ©AlphaMed Press.

Phenotypic high-throughput screening elucidates target pathway in breast cancer stem cell-like cells.

PubMed

Carmody, Leigh C; Germain, Andrew R; VerPlank, Lynn; Nag, Partha P; Muñoz, Benito; Perez, Jose R; Palmer, Michelle A J

2012-10-01

Cancer stem cells (CSCs) are resistant to standard cancer treatments and are likely responsible for cancer recurrence, but few therapies target this subpopulation. Due to the difficulty in propagating CSCs outside of the tumor environment, previous work identified CSC-like cells by inducing human breast epithelial cells into an epithelial-to-mesenchymal transdifferentiated state (HMLE_sh_ECad). A phenotypic screen was conducted against HMLE_sh_ECad with 300 718 compounds from the Molecular Libraries Small Molecule Repository to identify selective inhibitors of CSC growth. The screen yielded 2244 hits that were evaluated for toxicity and selectivity toward an isogenic control cell line. An acyl hydrazone scaffold emerged as a potent and selective scaffold targeting HMLE_sh_ECad. Fifty-three analogues were acquired and tested; compounds ranged in potency from 790 nM to inactive against HMLE_sh_ECad. Of the analogues, ML239 was best-in-class with an IC(50)= 1.18 µM against HMLE_sh_ECad, demonstrated a >23-fold selectivity over the control line, and was toxic to another CSC-like line, HMLE_shTwist, and a breast carcinoma cell line, MDA-MB-231. Gene expression studies conducted with ML239-treated cells showed altered gene expression in the NF-κB pathway in the HMLE_sh_ECad line but not in the isogenic control line. Future studies will be directed toward the identification of ML239 target(s).

Mesenchymal stem cells from adipose and bone marrow promote angiogenesis via distinct cytokine and protease expression mechanisms

PubMed Central

Kachgal, Suraj; Putnam, Andrew J.

2012-01-01

Using a fibrin-based angiogenesis model, we have established that there is no canonical mechanism used by ECs to degrade the surrounding extracellular matrix (ECM), but rather the set of proteases used is dependent on the mural cells providing the angiogenic cues. Mesenchymal stem cells (MSCs) originating from different tissues, which are thought to be phenotypically similar, promote angiogenesis through distinct mechanisms. Specifically, adipose-derived stem cells (ASCs) promote utilization of the plasminogen activator-plasmin axis by ECs as the primary means of vessel invasion and elongation in fibrin. Matrix metalloproteinases (MMPs) serve a purpose in regulating capillary diameter and possibly in stabilizing the nascent vessels. These proteolytic mechanisms are more akin to those involved in fibroblast-mediated angiogenesis than to those in bone marrow-derived stem cell (BMSC)-mediated angiogenesis. In addition, expression patterns of angiogenic factors such as urokinase plasminogen activator (uPA), hepatocyte growth factor (HGF), and tumor necrosis factor alpha (TNFα) were similar for ASC and fibroblast-mediated angiogenesis, and in direct contrast to BMSC-mediated angiogenesis. The present study illustrates that the nature of the heterotypic interactions between mural cells and endothelial cells depend on the identity of the mural cell used. Even MSCs which are shown to behave phenotypically similar do not stimulate angiogenesis via the same mechanisms. PMID:21104120

Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides.

PubMed

Rivera-Torres, Natalia; Banas, Kelly; Bialk, Pawel; Bloh, Kevin M; Kmiec, Eric B

2017-01-01

CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and CRISPR/Cas9 ribonucleoprotein complex.

Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides

PubMed Central

Rivera-Torres, Natalia; Bialk, Pawel; Bloh, Kevin M.; Kmiec, Eric B.

2017-01-01

CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and CRISPR/Cas9 ribonucleoprotein complex. PMID:28052104

Yersinia pestis targets neutrophils via complement receptor 3

PubMed Central

Merritt, Peter M.; Nero, Thomas; Bohman, Lesley; Felek, Suleyman; Krukonis, Eric S.; Marketon, Melanie M.

2015-01-01

Yersinia species display a tropism for lymphoid tissues during infection, and the bacteria select innate immune cells for delivery of cytotoxic effectors by the type III secretion system. Yet the mechanism for target cell selection remains a mystery. Here we investigate the interaction of Yersinia pestis with murine splenocytes to identify factors that participate in the targeting process. We find that interactions with primary immune cells rely on multiple factors. First, the bacterial adhesin Ail is required for efficient targeting of neutrophils in vivo. However, Ail does not appear to directly mediate binding to a specific cell type. Instead, we find that host serum factors direct Y. pestis to specific innate immune cells, particularly neutrophils. Importantly, specificity towards neutrophils was increased in the absence of bacterial adhesins due to reduced targeting of other cell types, but this phenotype was only visible in the presence of mouse serum. Addition of antibodies against complement receptor 3 and CD14 blocked target cell selection, suggesting that a combination of host factors participate in steering bacteria toward neutrophils during plague infection. PMID:25359083

Cell of Origin and Cancer Stem Cell Phenotype in Medulloblastomas

DTIC Science & Technology

2015-07-01

AWARD NUMBER: W81XWH-14-1-0115 TITLE: Cell of Origin and Cancer Stem Cell Phenotype in Medulloblastomas PRINCIPAL INVESTIGATOR: Kyuson Yun...YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE July 2015 2. REPORT TYPE Annual 3. DATES COVERED 1 Jul 2014 - 30 Jun 2015 4. TITLE AND SUBTITLE Cell ...of Origin and Cancer Stem Cell Phenotype in Medulloblastomas 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0115 5c. PROGRAM ELEMENT NUMBER 6

Theileria-transformed bovine leukocytes have cancer hallmarks.

PubMed

Tretina, Kyle; Gotia, Hanzel T; Mann, David J; Silva, Joana C

2015-07-01

The genus Theileria includes tick-transmitted apicomplexan parasites of ruminants with substantial economic impact in endemic countries. Some species, including Theileria parva and Theileria annulata, infect leukocytes where they induce phenotypes that are shared with some cancers, most notably immortalization, hyperproliferation, and dissemination. Despite considerable research into the affected host signaling pathways, the parasite proteins directly responsible for these host phenotypes remain unknown. In this review we outline current knowledge on the manipulation of host cells by transformation-inducing Theileria, and we propose that comparisons between cancer biology and host-Theileria interactions can reveal chemotherapeutic targets against Theileria-induced pathogenesis based on cancer treatment approaches. Copyright © 2015 Elsevier Ltd. All rights reserved.

Recent Advances in Microbial Single Cell Genomics Technology and Applications

NASA Astrophysics Data System (ADS)

Stepanauskas, R.

2016-02-01

Single cell genomics is increasingly utilized as a powerful tool to decipher the metabolic potential, evolutionary histories and in situ interactions of environmental microorganisms. This transformative technology recovers extensive information from cultivation-unbiased samples of individual, unicellular organisms. Thus, it does not require data binning into arbitrary phylogenetic or functional groups and therefore is highly compatible with agent-based modeling approaches. I will present several technological advances in this field, which significantly improve genomic data recovery from individual cells and provide direct linkages between cell's genomic and phenotypic properties. I will also demonstrate how these new technical capabilities help understanding the metabolic potential and viral infections of the "microbial dark matter" inhabiting aquatic and subsurface environments.

Interleukin-like EMT inducer regulates partial phenotype switching in MITF-low melanoma cell lines

PubMed Central

Noguchi, Ken; Dalton, Annamarie C.; Howley, Breege V.; McCall, Buckley J.; Yoshida, Akihiro; Diehl, J. Alan

2017-01-01

ILEI (FAM3C) is a secreted factor that contributes to the epithelial-to-mesenchymal transition (EMT), a cell biological process that confers metastatic properties to a tumor cell. Initially, we found that ILEI mRNA is highly expressed in melanoma metastases but not in primary tumors, suggesting that ILEI contributes to the malignant properties of melanoma. While melanoma is not an epithelial cell-derived tumor and does not undergo a traditional EMT, melanoma undergoes a similar process known as phenotype switching in which high (micropthalmia-related transcription factor) MITF expressing (MITF-high) proliferative cells switch to a low expressing (MITF-low) invasive state. We observed that MITF-high proliferative cells express low levels of ILEI (ILEI-low) and MITF-low invasive cells express high levels of ILEI (ILEI-high). We found that inducing phenotype switching towards the MITF-low invasive state increases ILEI mRNA expression, whereas phenotype switching towards the MITF-high proliferative state decreases ILEI mRNA expression. Next, we used in vitro assays to show that knockdown of ILEI attenuates invasive potential but not MITF expression or chemoresistance. Finally, we used gene expression analysis to show that ILEI regulates several genes involved in the MITF-low invasive phenotype including JARID1B, HIF-2α, and BDNF. Gene set enrichment analysis suggested that ILEI-regulated genes are enriched for JUN signaling, a known regulator of the MITF-low invasive phenotype. In conclusion, we demonstrate that phenotype switching regulates ILEI expression, and that ILEI regulates partial phenotype switching in MITF-low melanoma cell lines. PMID:28545079

Midbrain dopamine neurons reflect affiliation phenotypes in finches and are tightly coupled to courtship.

PubMed

Goodson, James L; Kabelik, David; Kelly, Aubrey M; Rinaldi, Jacob; Klatt, James D

2009-05-26

Mesolimbic dopamine (DA) circuits mediate a wide range of goal-oriented behavioral processes, and DA strongly influences appetitive and consummatory aspects of male sexual behavior. In both birds and mammals, mesolimbic projections arise primarily from the ventral tegmental area (VTA), with a smaller contribution from the midbrain central gray (CG). Despite the well known importance of the VTA cell group for incentive motivation functions, relationships of VTA subpopulations to specific aspects of social phenotype remain wholly undescribed. We now show that in male zebra finches (Estrildidae: Taeniopygia guttata), Fos activity within a subpopulation of tyrosine hydroxylase-immunoreactive (TH-ir; presumably dopaminergic) neurons in the caudal VTA is significantly correlated with courtship singing and coupled to gonadal state. In addition, the number of TH-ir neurons in this caudal subpopulation dichotomously differentiates courting from non-courting male phenotypes, and evolves in relation to sociality (flocking vs. territorial) across several related finch species. Combined, these findings for the VTA suggest that divergent social phenotypes may arise due to the differential assignment of "incentive value" to conspecific stimuli. TH-ir neurons of the CG (a population of unknown function in mammals) exhibit properties that are even more selectively and tightly coupled to the expression of courtship phenotypes (and appetitive courtship singing), both in terms of TH-ir cell number, which correlates significantly with constitutive levels of courtship motivation, and with TH-Fos colocalization, which increases in direct proportion to the phasic expression of song. We propose that these neurons may be core components of social communication circuits across diverse vertebrate taxa.

Cell population structure prior to bifurcation predicts efficiency of directed differentiation in human induced pluripotent cells

PubMed Central

Bargaje, Rhishikesh; Trachana, Kalliopi; Shelton, Martin N.; McGinnis, Christopher S.; Zhou, Joseph X.; Chadick, Cora; Cook, Savannah; Cavanaugh, Christopher; Huang, Sui; Hood, Leroy

2017-01-01

Steering the differentiation of induced pluripotent stem cells (iPSCs) toward specific cell types is crucial for patient-specific disease modeling and drug testing. This effort requires the capacity to predict and control when and how multipotent progenitor cells commit to the desired cell fate. Cell fate commitment represents a critical state transition or “tipping point” at which complex systems undergo a sudden qualitative shift. To characterize such transitions during iPSC to cardiomyocyte differentiation, we analyzed the gene expression patterns of 96 developmental genes at single-cell resolution. We identified a bifurcation event early in the trajectory when a primitive streak-like cell population segregated into the mesodermal and endodermal lineages. Before this branching point, we could detect the signature of an imminent critical transition: increase in cell heterogeneity and coordination of gene expression. Correlation analysis of gene expression profiles at the tipping point indicates transcription factors that drive the state transition toward each alternative cell fate and their relationships with specific phenotypic readouts. The latter helps us to facilitate small molecule screening for differentiation efficiency. To this end, we set up an analysis of cell population structure at the tipping point after systematic variation of the protocol to bias the differentiation toward mesodermal or endodermal cell lineage. We were able to predict the proportion of cardiomyocytes many days before cells manifest the differentiated phenotype. The analysis of cell populations undergoing a critical state transition thus affords a tool to forecast cell fate outcomes and can be used to optimize differentiation protocols to obtain desired cell populations. PMID:28167799

Cell population structure prior to bifurcation predicts efficiency of directed differentiation in human induced pluripotent cells.

PubMed

Bargaje, Rhishikesh; Trachana, Kalliopi; Shelton, Martin N; McGinnis, Christopher S; Zhou, Joseph X; Chadick, Cora; Cook, Savannah; Cavanaugh, Christopher; Huang, Sui; Hood, Leroy

2017-02-28

Steering the differentiation of induced pluripotent stem cells (iPSCs) toward specific cell types is crucial for patient-specific disease modeling and drug testing. This effort requires the capacity to predict and control when and how multipotent progenitor cells commit to the desired cell fate. Cell fate commitment represents a critical state transition or "tipping point" at which complex systems undergo a sudden qualitative shift. To characterize such transitions during iPSC to cardiomyocyte differentiation, we analyzed the gene expression patterns of 96 developmental genes at single-cell resolution. We identified a bifurcation event early in the trajectory when a primitive streak-like cell population segregated into the mesodermal and endodermal lineages. Before this branching point, we could detect the signature of an imminent critical transition: increase in cell heterogeneity and coordination of gene expression. Correlation analysis of gene expression profiles at the tipping point indicates transcription factors that drive the state transition toward each alternative cell fate and their relationships with specific phenotypic readouts. The latter helps us to facilitate small molecule screening for differentiation efficiency. To this end, we set up an analysis of cell population structure at the tipping point after systematic variation of the protocol to bias the differentiation toward mesodermal or endodermal cell lineage. We were able to predict the proportion of cardiomyocytes many days before cells manifest the differentiated phenotype. The analysis of cell populations undergoing a critical state transition thus affords a tool to forecast cell fate outcomes and can be used to optimize differentiation protocols to obtain desired cell populations.

Isolation, characterization and recombinant protein expression in Veggie-CHO: A serum-free CHO host cell line.

PubMed

Rasmussen, B; Davis, R; Thomas, J; Reddy, P

1998-11-01

The dihydrofolate reductase-deficient Chinese hamster ovary cell line, DXB11-CHO, commonly used as a host cell for the production of recombinant proteins requires 7.5% serum-supplementation for optimal growth. Regulatory issues surrounding the use of serum in clinical production processes and the direct and indirect costs of using serum in large-scale production and recovery processes have triggered efforts to derive serum-independent host cell lines. We have successfully isolated a serum-free host that we named Veggie- CHO. Veggie-CHO was generated by adapting DXB11-CHO cells to growth in serum-free media in the absence of exogenous growth factors such as Transferrin and Insulin-like growth factor, which we have previously shown to be essential for growth and viability of DXB11- CHO cells. Veggie-CHO cells have been shown to maintain an average doubling time of 22 hr in continuous growth cultures over a period of three months and have retained the dihydrofolate reductase -deficient phenotype of their parental DXB11-CHO cells. These properties and the stability of its serum-free phenotype have allowed the use of Veggie- CHO as host cells for transfection and amplified expression of recombinant proteins. We describe the derivation a serum-free recombinant cell line with an average doubling time of 20 hr and specific productivity of 2.5 Units recombinant Flt-3L protein per 10e6 cells per day.

Single cells from human primary colorectal tumors exhibit polyfunctional heterogeneity in secretions of ELR+ CXC chemokines.

PubMed

Adalsteinsson, Viktor A; Tahirova, Narmin; Tallapragada, Naren; Yao, Xiaosai; Campion, Liam; Angelini, Alessandro; Douce, Thomas B; Huang, Cindy; Bowman, Brittany; Williamson, Christina A; Kwon, Douglas S; Wittrup, K Dane; Love, J Christopher

2013-10-01

Cancer is an inflammatory disease of tissue that is largely influenced by the interactions between multiple cell types, secreted factors, and signal transduction pathways. While single-cell sequencing continues to refine our understanding of the clonotypic heterogeneity within tumors, the complex interplay between genetic variations and non-genetic factors ultimately affects therapeutic outcome. Much has been learned through bulk studies of secreted factors in the tumor microenvironment, but the secretory behavior of single cells has been largely uncharacterized. Here we directly profiled the secretions of ELR+ CXC chemokines from thousands of single colorectal tumor and stromal cells, using an array of subnanoliter wells and a technique called microengraving to characterize both the rates of secretion of several factors at once and the numbers of cells secreting each chemokine. The ELR+ CXC chemokines are highly redundant, pro-angiogenic cytokines that signal via the CXCR1 and CXCR2 receptors, influencing tumor growth and progression. We find that human primary colorectal tumor and stromal cells exhibit polyfunctional heterogeneity in the combinations and magnitudes of secretions for these chemokines. In cell lines, we observe similar variance: phenotypes observed in bulk can be largely absent among the majority of single cells, and discordances exist between secretory states measured and gene expression for these chemokines among single cells. Together, these measures suggest secretory states among tumor cells are complex and can evolve dynamically. Most importantly, this study reveals new insight into the intratumoral phenotypic heterogeneity of human primary tumors.

Phenotype and function of CD209+ bovine blood dendritic cells, monocyte-derived-dendritic cells and monocyte-derived macrophages

USDA-ARS?s Scientific Manuscript database

Phylogenic comparisons of the mononuclear phagocyte system (MPS) of humans and mice demonstrate phenotypic divergence of dendritic cell (DC) subsets that play similar roles in innate and adaptive immunity. Although differing in phenotype, DC can be classified into four groups according to ontogeny a...

PC12 Cells Differentiate into Chromaffin Cell-Like Phenotype in Coculture with Adrenal Medullary Endothelial Cells

NASA Astrophysics Data System (ADS)

Mizrachi, Yaffa; Naranjo, Jose R.; Levi, Ben-Zion; Pollard, Harvey B.; Lelkes, Peter I.

1990-08-01

Previously we described specific in vitro interactions between PC12 cells, a cloned, catecholamine-secreting pheochromocytoma cell line derived from the rat adrenal medulla, and bovine adrenal medullary endothelial cells. We now demonstrate that these interactions induce the PC12 cells to acquire physical and biochemical characteristics reminiscent of chromaffin cells. Under coculture conditions involving direct cell-cell contact, the endothelial cells and the PC12 cells reduced their rates of proliferation; upon prolonged coculture PC12 cells clustered into nests of cells similar to the organization of chromaffin cells seen in vivo. Within 3 days in coculture with endothelial cells, but not with unrelated control cells, PC12 cells synthesized increased levels of [Met]enkephalin. In addition, PC12 cells, growing on confluent endothelial monolayers, failed to extend neurites in response to nerve growth factor. Neither medium conditioned by endothelial cells nor fixed endothelial cells could by themselves induce all of these different phenomena in the PC12 cells. These results suggest that under coculture conditions PC12 cells change their state of differentiation toward a chromaffin cell-like phenotype. The rapid, transient increase in the expression of the protooncogene c-fos suggests that the mechanism(s) inducing the change in the state of differentiation in PC12 cells in coculture with the endothelial cells may be distinct from that described for the differentiation of PC12 cells--e.g., by glucocorticoids. We propose that similar interactions between endothelial cells and chromaffin cell precursors may occur during embryonic development and that these interactions might be instrumental for the organ-specific differentiation of the adrenal medulla in vivo.

Demonstration of intermediate cells during human prostate epithelial differentiation in situ and in vitro using triple-staining confocal scanning microscopy.

PubMed

van Leenders, G; Dijkman, H; Hulsbergen-van de Kaa, C; Ruiter, D; Schalken, J

2000-08-01

In human prostate epithelium, morphologically basal and luminal cells can be discriminated. The basal cell layer that putatively contains progenitor cells of the secretory epithelium is characterized by the expression of keratins (K) 5 and 14. Luminal cells represent the secretory compartment of the epithelium and express K8 and 18. We developed a technique for the simultaneous analysis of K5, 14, and 18 to identify intermediate cell stages in the prostate epithelium and to study the dynamic aspects of its differentiation in vitro. Nonmalignant prostate tissue and primary epithelial cultures were immunohistochemically characterized using triple staining with antibodies for K5, K14, and K18. Antibodies for K18 and K5 were conjugated directly with fluorochromes Alexa 488 and 546. K14 was visualized indirectly with streptavidin-Cy5. Keratin expression was analyzed by confocal scanning microscopy. The occurrence of exocrine and neuroendocrine differentiation in culture was determined via antibodies to prostate-specific antigen (PSA), chromogranin A, and serotonin. We found that basal cells expressed either K5(++)/14(++)/18+ or K5(++)/18+. The majority of luminal cells expressed K18(++), but colocalization of K5+/18(++) were recognized. Epithelial monolayer cultures predominantly revealed the basal cell phenotype K5(++)/14(++)/18+, whereas intermediate subpopulations expressing K5+/14+/18(++) and K5+/18(++) were also identified. On confluence, differentiation was induced as multicellular gland-like buds, and extensions became evident on top of the monolayer. These structures were composed of K18(++)- and K5+/18(+)-positive cell clusters surrounded by phenotypically basal cells. Few multicellular structures and cells in the monolayer showed exocrine differentiation (PSA+), but expression of chromogranin A and serotonin was absent. We conclude that simultaneous evaluation of keratin expression is useful for analyzing epithelial differentiation in the prostate. During this process, putative stem cells phenotypically resembling K5(++)/14(++)/18+ differentiate toward luminal cells (K18(++)) via intermediate cell stages, as identified by up-regulation of K18 and down-regulation of K5 and 14.

Early transduction produces highly functional chimeric antigen receptor-modified virus-specific T-cells with central memory markers: a Production Assistant for Cell Therapy (PACT) translational application.

PubMed

Sun, Jiali; Huye, Leslie E; Lapteva, Natalia; Mamonkin, Maksim; Hiregange, Manasa; Ballard, Brandon; Dakhova, Olga; Raghavan, Darshana; Durett, April G; Perna, Serena K; Omer, Bilal; Rollins, Lisa A; Leen, Ann M; Vera, Juan F; Dotti, Gianpietro; Gee, Adrian P; Brenner, Malcolm K; Myers, Douglas G; Rooney, Cliona M

2015-01-01

Virus-specific T-cells (VSTs) proliferate exponentially after adoptive transfer into hematopoietic stem cell transplant (HSCT) recipients, eliminate virus infections, then persist and provide long-term protection from viral disease. If VSTs behaved similarly when modified with tumor-specific chimeric antigen receptors (CARs), they should have potent anti-tumor activity. This theory was evaluated by Cruz et al. in a previous clinical trial with CD19.CAR-modified VSTs, but there was little apparent expansion of these cells in patients. In that study, VSTs were gene-modified on day 19 of culture and we hypothesized that by this time, sufficient T-cell differentiation may have occurred to limit the subsequent proliferative capacity of the transduced T-cells. To facilitate the clinical testing of this hypothesis in a project supported by the NHLBI-PACT mechanism, we developed and optimized a good manufacturing practices (GMP) compliant method for the early transduction of VSTs directed to Epstein-Barr virus (EBV), Adenovirus (AdV) and cytomegalovirus (CMV) using a CAR directed to the tumor-associated antigen disialoganglioside (GD2). Ad-CMVpp65-transduced EBV-LCLs effectively stimulated VSTs directed to all three viruses (triVSTs). Transduction efficiency on day three was increased in the presence of cytokines and high-speed centrifugation of retroviral supernatant onto retronectin-coated plates, so that under optimal conditions up to 88% of tetramer-positive VSTs expressed the GD2.CAR. The average transduction efficiency of early-and late transduced VSTs was 55 ± 4% and 22 ± 5% respectively, and early-transduced VSTs maintained higher frequencies of T cells with central memory or intermediate memory phenotypes. Early-transduced VSTs also had higher proliferative capacity and produced higher levels of TH1 cytokines IL-2, TNF-α, IFN-γ, MIP-1α, MIP-1β and other cytokines in vitro. We developed a rapid and GMP compliant method for the early transduction of multivirus-specific T-cells that allowed stable expression of high levels of a tumor directed CAR. Since a proportion of early-transduced CAR-VSTs had a central memory phenotype, they should expand and persist in vivo, simultaneously protecting against infection and targeting residual malignancy. This manufacturing strategy is currently under clinical investigation in patients receiving allogeneic HSCT for relapsed neuroblastoma and B-cell malignancies (NCT01460901 using a GD2.CAR and NCT00840853 using a CD19.CAR).

Endothelial cells respond to the direction of mechanical stimuli through SMAD signaling to regulate coronary artery size.

PubMed

Poduri, Aruna; Chang, Andrew H; Raftrey, Brian; Rhee, Siyeon; Van, Mike; Red-Horse, Kristy

2017-09-15

How mechanotransduction intersects with chemical and transcriptional factors to shape organogenesis is an important question in developmental biology. This is particularly relevant to the cardiovascular system, which uses mechanical signals from flowing blood to stimulate cytoskeletal and transcriptional responses that form a highly efficient vascular network. Using this system, artery size and structure are tightly regulated, but the underlying mechanisms are poorly understood. Here, we demonstrate that deletion of Smad4 increased the diameter of coronary arteries during mouse embryonic development, a phenotype that followed the initiation of blood flow. At the same time, the BMP signal transducers SMAD1/5/8 were activated in developing coronary arteries. In a culture model of blood flow-induced shear stress, human coronary artery endothelial cells failed to align when either BMPs were inhibited or SMAD4 was depleted. In contrast to control cells, SMAD4- deficient cells did not migrate against the direction of shear stress and increased proliferation rates specifically under flow. Similar alterations were seen in coronary arteries in vivo Thus, endothelial cells perceive the direction of blood flow and respond through SMAD signaling to regulate artery size. © 2017. Published by The Company of Biologists Ltd.

In situ elasticity modulation with dynamic substrates to direct cell phenotype

PubMed Central

Kloxin, April M.; Benton, Julie A.; Anseth, Kristi S.

2009-01-01

Microenvironment elasticity influences critical cell functions such as differentiation, cytoskeletal organization, and process extension. Unfortunately, few materials allow elasticity modulation in real-time to probe its direct effect on these dynamic cellular processes. Here, a new approach is presented for the photochemical modulation of elasticity within the cell's microenvironment at any point in time. A photodegradable hydrogel was irradiated and degraded under cytocompatible conditions to generate a wide range of elastic moduli similar to soft tissues and characterized using rheometry and atomic force microscopy (AFM). The effect of the elastic modulus on valvular interstitial cell (VIC) activation into myofibroblasts was explored. In these studies, gradient samples were used to identify moduli that either promote or suppress VIC myofibroblastic activation. With this knowledge, VICs were cultured on a high modulus, activating hydrogel substrate, and uniquely, results show that decreasing the substrate modulus with irradiation reverses this activation, demonstrating that myofibroblasts can be de-activated solely by changing the modulus of the underlying substrate. This finding is important for the rational design of biomaterials for tissue regeneration and offers insight into fibrotic disease progression. These photodegradable hydrogels demonstrate the capability to both probe and direct cell function through dynamic changes in substrate elasticity. PMID:19788947

Innate Lymphoid Cells: a new paradigm in immunology

PubMed Central

Eberl, Gérard; Colonna, Marco; Di Santo, James P.; McKenzie, Andrew N.J.

2016-01-01

Summary Innate lymphoid cells (ILCs) are a growing family of immune cells that mirror the phenotypes and functions of T cells. However, in contrast to T cells, ILCs do not express acquired antigen receptors or undergo clonal selection and expansion when stimulated. Instead, ILCs react promptly to signals from infected or injured tissues and produce an array of secreted proteins termed cytokines that direct the developing immune response into one that is adapted to the original insult. The complex crosstalk between microenvironment, ILCs and adaptive immunity remains to be fully deciphered. Only by understanding these complex regulatory networks can the power of ILCs be controlled or unleashed to regulate or enhance immune responses in disease prevention and therapy. PMID:25999512

Hoxb4 overexpression in CD4 memory phenotype T cells increases the central memory population upon homeostatic proliferation.

PubMed

Frison, Héloïse; Giono, Gloria; Thébault, Paméla; Fournier, Marilaine; Labrecque, Nathalie; Bijl, Janet J

2013-01-01

Memory T cell populations allow a rapid immune response to pathogens that have been previously encountered and thus form the basis of success in vaccinations. However, the molecular pathways underlying the development and maintenance of these cells are only starting to be unveiled. Memory T cells have the capacity to self renew as do hematopoietic stem cells, and overlapping gene expression profiles suggested that these cells might use the same self-renewal pathways. The transcription factor Hoxb4 has been shown to promote self-renewal divisions of hematopoietic stem cells resulting in an expansion of these cells. In this study we investigated whether overexpression of Hoxb4 could provide an advantage to CD4 memory phenotype T cells in engrafting the niche of T cell deficient mice following adoptive transfer. Competitive transplantation experiments demonstrated that CD4 memory phenotype T cells derived from mice transgenic for Hoxb4 contributed overall less to the repopulation of the lymphoid organs than wild type CD4 memory phenotype T cells after two months. These proportions were relatively maintained following serial transplantation in secondary and tertiary mice. Interestingly, a significantly higher percentage of the Hoxb4 CD4 memory phenotype T cell population expressed the CD62L and Ly6C surface markers, characteristic for central memory T cells, after homeostatic proliferation. Thus Hoxb4 favours the maintenance and increase of the CD4 central memory phenotype T cell population. These cells are more stem cell like and might eventually lead to an advantage of Hoxb4 T cells after subjecting the cells to additional rounds of proliferation.

Directional selection effects on patterns of phenotypic (co)variation in wild populations.

PubMed

Assis, A P A; Patton, J L; Hubbe, A; Marroig, G

2016-11-30

Phenotypic (co)variation is a prerequisite for evolutionary change, and understanding how (co)variation evolves is of crucial importance to the biological sciences. Theoretical models predict that under directional selection, phenotypic (co)variation should evolve in step with the underlying adaptive landscape, increasing the degree of correlation among co-selected traits as well as the amount of genetic variance in the direction of selection. Whether either of these outcomes occurs in natural populations is an open question and thus an important gap in evolutionary theory. Here, we documented changes in the phenotypic (co)variation structure in two separate natural populations in each of two chipmunk species (Tamias alpinus and T. speciosus) undergoing directional selection. In populations where selection was strongest (those of T. alpinus), we observed changes, at least for one population, in phenotypic (co)variation that matched theoretical expectations, namely an increase of both phenotypic integration and (co)variance in the direction of selection and a re-alignment of the major axis of variation with the selection gradient. © 2016 The Author(s).

GLI1 inhibition promotes epithelial-to-mesenchymal transition in pancreatic cancer cells

PubMed Central

Joost, Simon; Almada, Luciana L.; Rohnalter, Verena; Holz, Philipp S.; Vrabel, Anne M.; Fernandez-Barrena, Maite G.; McWilliams, Robert R.; Krause, Michael; Fernandez-Zapico, Martin E.; Lauth, Matthias

2011-01-01

The Hedgehog (HH) pathway has been identified as an important deregulated signal transduction pathway in pancreatic ductal adenocarcinoma (PDAC), a cancer type characterized by a highly metastatic phenotype. In PDAC, the canonical HH pathway activity is restricted to the stromal compartment while HH signaling in the tumor cells is reduced as a consequence of constitutive KRAS activation. Here we report that in the tumor compartment of PDAC the HH pathway effector transcription factor GLI1 regulates epithelial differentiation. RNAi-mediated knockdown of GLI1 abolished characteristics of epithelial differentiation, increased cell motility and synergized with TGFβ to induce an epithelial-to-mesenchymal transition (EMT). Notably, EMT conversion in PDAC cells occurred in the absence of induction of SNAIL or SLUG, two canonical inducers of EMT in many other settings. Further mechanistic analysis revealed that GLI1 directly regulated the transcription of E-cadherin, a key determinant of epithelial tissue organization. Collectively, our findings identify GLI1 as an important positive regulator of epithelial differentiation, and they offer an explanation for how decreased levels of GLI1 are likely to contribute to the highly metastatic phenotype of PDAC. PMID:22086851

Disease signatures for schizophrenia and bipolar disorder using patient-derived induced pluripotent stem cells.

PubMed

Watmuff, Bradley; Berkovitch, Shaunna S; Huang, Joanne H; Iaconelli, Jonathan; Toffel, Steven; Karmacharya, Rakesh

2016-06-01

Schizophrenia and bipolar disorder are complex psychiatric disorders that present unique challenges in the study of disease biology. There are no objective biological phenotypes for these disorders, which are characterized by complex genetics and prominent roles for gene-environment interactions. The study of the neurobiology underlying these severe psychiatric disorders has been hindered by the lack of access to the tissue of interest - neurons from patients. The advent of reprogramming methods that enable generation of induced pluripotent stem cells (iPSCs) from patient fibroblasts and peripheral blood mononuclear cells has opened possibilities for new approaches to study relevant disease biology using iPSC-derived neurons. While early studies with patient iPSCs have led to promising and intriguing leads, significant hurdles remain in our attempts to capture the complexity of these disorders in vitro. We present here an overview of studies to date of schizophrenia and bipolar disorder using iPSC-derived neuronal cells and discuss potential future directions that can result in the identification of robust and valid cellular phenotypes that in turn can lay the groundwork for meaningful clinical advances. Copyright © 2016 Elsevier Inc. All rights reserved.

The effect of hydrodynamic conditions on the phenotype of Pseudomonas fluorescens biofilms.

PubMed

Simões, Manuel; Pereira, Maria O; Sillankorva, Sanna; Azeredo, Joana; Vieira, Maria J

2007-01-01

This study investigated the phenotypic characteristics of monoculture P. fluorescens biofilms grown under turbulent and laminar flow, using flow cells reactors with stainless steel substrata. The cellular physiology and the overall biofilm activity, structure and composition were characterized, and compared, within hydrodynamically distinct conditions. The results indicate that turbulent flow-generated biofilm cells were significantly less extensive, with decreased metabolic activity and a lower protein and polysaccharides composition per cell than those from laminar flow-generated biofilms. The effect of flow regime did not cause significantly different outer membrane protein expression. From the analysis of biofilm activity, structure and composition, turbulent flow-generated biofilms were metabolically more active, had twice more mass per cm(2), and higher cellular density and protein content (mainly cellular) than laminar flow-generated biofilms. Conversely, laminar flow-generated biofilms presented higher total and matrix polysaccharide contents. Direct visualisation and scanning electron microscopy analysis showed that these different flows generate structurally different biofilms, corroborating the quantitative results. The combination of applied methods provided useful information regarding a broad spectrum of biofilm parameters, which can contribute to control and model biofilm processes.

Seedling lethality in Nicotiana plumbaginifolia conferred by Ds transposable element insertion into a plant-specific gene.

PubMed

Majira, Amel; Domin, Monique; Grandjean, Olivier; Gofron, Krystyna; Houba-Hérin, Nicole

2002-10-01

A seedling lethal mutant of Nicotiana plumbaginifolia (sdl-1) was isolated by transposon tagging using a maize Dissociation (Ds) element. The insertion mutation was produced by direct co-transformation of protoplasts with two plasmids: one containing Ds and a second with an Ac transposase gene. sdl-1 seedlings exhibit several phenotypes: swollen organs, short hypocotyls in light and dark conditions, and enlarged and multinucleated cells, that altogether suggest cell growth defects. Mutant cells are able to proliferate under in vitro culture conditions. Genomic DNA sequences bordering the transposon were used to recover cDNA from the normal allele. Complementation of the mutant phenotype with the cDNA confirmed that the transposon had caused the mutation. The Ds element was inserted into the first exon of the open reading frame and the homozygous mutant lacked detectable transcript. Phenocopies of the mutant were obtained by an antisense approach. SDL-1 encodes a novel protein found in several plant genomes but apparently missingfrom animal and fungal genomes; the protein is highly conserved and has a potential plastid targeting motif.

Neural Stem Cells Derived Directly from Adipose Tissue.

PubMed

Petersen, Eric D; Zenchak, Jessica R; Lossia, Olivia V; Hochgeschwender, Ute

2018-05-01

Neural stem cells (NSCs) are characterized as self-renewing cell populations with the ability to differentiate into the multiple tissue types of the central nervous system. These cells can differentiate into mature neurons, astrocytes, and oligodendrocytes. This category of stem cells has been shown to be a promisingly effective treatment for neurodegenerative diseases and neuronal injury. Most treatment studies with NSCs in animal models use embryonic brain-derived NSCs. This approach presents both ethical and feasibility issues for translation to human patients. Adult tissue is a more practical source of stem cells for transplantation therapies in humans. Some adult tissues such as adipose tissue and bone marrow contain a wide variety of stem cell populations, some of which have been shown to be similar to embryonic stem cells, possessing many pluripotent properties. Of these stem cell populations, some are able to respond to neuronal growth factors and can be expanded in vitro, forming neurospheres analogous to cells harvested from embryonic brain tissue. In this study, we describe a method for the collection and culture of cells from adipose tissue that directly, without going through intermediates such as mesenchymal stem cells, results in a population of NSCs that are able to be expanded in vitro and be differentiated into functional neuronal cells. These adipose-derived NSCs display a similar phenotype to those directly derived from embryonic brain. When differentiated into neurons, cells derived from adipose tissue have spontaneous spiking activity with network characteristics similar to that of neuronal cultures.

Lineage Tracking for Probing Heritable Phenotypes at Single-Cell Resolution

PubMed Central

Cottinet, Denis; Condamine, Florence; Bremond, Nicolas; Griffiths, Andrew D.; Rainey, Paul B.; de Visser, J. Arjan G. M.; Baudry, Jean; Bibette, Jérôme

2016-01-01

Determining the phenotype and genotype of single cells is central to understand microbial evolution. DNA sequencing technologies allow the detection of mutants at high resolution, but similar approaches for phenotypic analyses are still lacking. We show that a drop-based millifluidic system enables the detection of heritable phenotypic changes in evolving bacterial populations. At time intervals, cells were sampled and individually compartmentalized in 100 nL drops. Growth through 15 generations was monitored using a fluorescent protein reporter. Amplification of heritable changes–via growth–over multiple generations yields phenotypically distinct clusters reflecting variation relevant for evolution. To demonstrate the utility of this approach, we follow the evolution of Escherichia coli populations during 30 days of starvation. Phenotypic diversity was observed to rapidly increase upon starvation with the emergence of heritable phenotypes. Mutations corresponding to each phenotypic class were identified by DNA sequencing. This scalable lineage-tracking technology opens the door to large-scale phenotyping methods with special utility for microbiology and microbial population biology. PMID:27077662

Lineage Tracking for Probing Heritable Phenotypes at Single-Cell Resolution.

PubMed

Cottinet, Denis; Condamine, Florence; Bremond, Nicolas; Griffiths, Andrew D; Rainey, Paul B; de Visser, J Arjan G M; Baudry, Jean; Bibette, Jérôme

2016-01-01

Determining the phenotype and genotype of single cells is central to understand microbial evolution. DNA sequencing technologies allow the detection of mutants at high resolution, but similar approaches for phenotypic analyses are still lacking. We show that a drop-based millifluidic system enables the detection of heritable phenotypic changes in evolving bacterial populations. At time intervals, cells were sampled and individually compartmentalized in 100 nL drops. Growth through 15 generations was monitored using a fluorescent protein reporter. Amplification of heritable changes-via growth-over multiple generations yields phenotypically distinct clusters reflecting variation relevant for evolution. To demonstrate the utility of this approach, we follow the evolution of Escherichia coli populations during 30 days of starvation. Phenotypic diversity was observed to rapidly increase upon starvation with the emergence of heritable phenotypes. Mutations corresponding to each phenotypic class were identified by DNA sequencing. This scalable lineage-tracking technology opens the door to large-scale phenotyping methods with special utility for microbiology and microbial population biology.

Antibacterial Compounds of Canadian Honeys Target Bacterial Cell Wall Inducing Phenotype Changes, Growth Inhibition and Cell Lysis That Resemble Action of β-Lactam Antibiotics

PubMed Central

Brudzynski, Katrina; Sjaarda, Calvin

2014-01-01

Honeys show a desirable broad spectrum activity against Gram-positive and negative bacteria making antibacterial activity an intrinsic property of honey and a desirable source for new drug development. The cellular targets and underlying mechanism of action of honey antibacterial compounds remain largely unknown. To facilitate the target discovery, we employed a method of phenotypic profiling by directly comparing morphological changes in Escherichia coli induced by honeys to that of ampicillin, the cell wall-active β-lactam of known mechanism of action. Firstly, we demonstrated the purity of tested honeys from potential β-lactam contaminations using quantitative LC-ESI-MS. Exposure of log-phase E. coli to honey or ampicillin resulted in time- and concentration-dependent changes in bacterial cell shape with the appearance of filamentous phenotypes at sub-inhibitory concentrations and spheroplasts at the MBC. Cell wall destruction by both agents, clearly visible on microscopic micrographs, was accompanied by increased permeability of the lipopolysaccharide outer membrane as indicated by fluorescence-activated cell sorting (FACS). More than 90% E. coli exposed to honey or ampicillin became permeable to propidium iodide. Consistently with the FACS results, both honey-treated and ampicillin-treated E. coli cells released lipopolysaccharide endotoxins at comparable levels, which were significantly higher than controls (p<0.0001). E. coli cells transformed with the ampicillin-resistance gene (β–lactamase) remained sensitive to honey, displayed the same level of cytotoxicity, cell shape changes and endotoxin release as ampicillin-sensitive cells. As expected, β–lactamase protected the host cell from antibacterial action of ampicillin. Thus, both honey and ampicillin induced similar structural changes to the cell wall and LPS and that this ability underlies antibacterial activities of both agents. Since the cell wall is critical for cell growth and survival, honey active compounds would be highly applicable for therapeutic purposes while differences in the mode of action between honey and ampicillin may provide clinical advantage in eradicating β-lactam-resistant pathogens. PMID:25191847

Implications of the Hybrid Epithelial/Mesenchymal Phenotype in Metastasis

PubMed Central

Jolly, Mohit Kumar; Boareto, Marcelo; Huang, Bin; Jia, Dongya; Lu, Mingyang; Ben-Jacob, Eshel; Onuchic, José N.; Levine, Herbert

2015-01-01

Transitions between epithelial and mesenchymal phenotypes – the epithelial to ­mesenchymal transition (EMT) and its reverse the mesenchymal to epithelial transition (MET) – are hallmarks of cancer metastasis. While transitioning between the epithelial and mesenchymal phenotypes, cells can also attain a hybrid epithelial/mesenchymal (E/M) (i.e., partial or intermediate EMT) phenotype. Cells in this phenotype have mixed epithelial (e.g., adhesion) and mesenchymal (e.g., migration) properties, thereby allowing them to move collectively as clusters. If these clusters reach the bloodstream intact, they can give rise to clusters of circulating tumor cells (CTCs), as have often been seen experimentally. Here, we review the operating principles of the core regulatory network for EMT/MET that acts as a “three-way” switch giving rise to three distinct phenotypes – E, M and hybrid E/M – and present a theoretical framework that can elucidate the role of many other players in regulating epithelial plasticity. Furthermore, we highlight recent studies on partial EMT and its association with drug resistance and tumor-initiating potential; and discuss how cell–cell communication between cells in a partial EMT phenotype can enable the formation of clusters of CTCs. These clusters can be more apoptosis-resistant and have more tumor-initiating potential than singly moving CTCs with a wholly mesenchymal (complete EMT) phenotype. Also, more such clusters can be formed under inflammatory conditions that are often generated by various therapies. Finally, we discuss the multiple advantages that the partial EMT or hybrid E/M phenotype have as compared to a complete EMT phenotype and argue that these collectively migrating cells are the primary “bad actors” of metastasis. PMID:26258068

Genome-Wide Search Reveals the Existence of a Limited Number of Thyroid Hormone Receptor Alpha Target Genes in Cerebellar Neurons

PubMed Central

Chatonnet, Fabrice; Guyot, Romain; Picou, Frédéric; Bondesson, Maria; Flamant, Frederic

2012-01-01

Thyroid hormone (T3) has a major influence on cerebellum post-natal development. The major phenotypic landmark of exposure to low levels of T3 during development (hypothyroidism) in the cerebellum is the retarded inward migration of the most numerous cell type, granular neurons. In order to identify the direct genetic regulation exerted by T3 on cerebellar neurons and their precursors, we used microarray RNA hybridization to perform a time course analysis of T3 induced gene expression in primary cultures of cerebellar neuronal cell. These experiments suggest that we identified a small set of genes which are directly regulated, both in vivo and in vitro, during cerebellum post-natal development. These modest changes suggest that T3 does not acts directly on granular neurons and mainly indirectly influences the cellular interactions taking place during development. PMID:22586439

Allogeneic substitution for nominal antigen-specific T-cell clone reactivity in schistosomiasis.

PubMed Central

Linette, G P; Lammie, P J; Phillips, S M

1986-01-01

The present studies have established the nature of a T-cell clone which demonstrates dual reactivity directed against Schistosoma mansoni antigen presented by syngeneic antigen presenting cells and against allogeneic cells. Clone G4, when stimulated by either antigen (SEA) or allogeneic cells (PL/J), exhibits similar functional and phenotypic characteristics. A subclone of G4, G4A.1, which has been maintained in continuous mixed lymphocyte culture for 12 months (in the absence of SEA), retains comparable reactivity with respect to proliferation and ability to produce lymphokines, transfer delayed-type hypersensitivity, and produce in vitro granulomas in response to SEA. Normal antigenic stimulation is highly contingent upon I-Ab compatibility while antibody blocking experiments map allo-reactivity to I-Eu. The failure of B10.PL spleen cells to stimulate G4, however, suggests that alloreactivity may be directed against the recently described Mls X locus. Both allogeneic and nominal antigen induced T-cell activation are blocked by antibody directed against L3T4A, confirming Class II MHC restriction for both types of stimulation. These studies suggest that stimulation of T cells by either alloantigen or nominal antigen elicits qualitatively similar functional profiles, and further suggest the feasibility of producing large numbers of nominal antigen reactive cloned T cells in the absence of nominal antigen under mixed lymphocyte culture conditions. PMID:2420707

Resolving Salmonella infection reveals dynamic and persisting changes in murine bone marrow progenitor cell phenotype and function

PubMed Central

Ross, Ewan A; Flores-Langarica, Adriana; Bobat, Saeeda; Coughlan, Ruth E; Marshall, Jennifer L; Hitchcock, Jessica R; Cook, Charlotte N; Carvalho-Gaspar, Manuela M; Mitchell, Andrea M; Clarke, Mary; Garcia, Paloma; Cobbold, Mark; Mitchell, Tim J; Henderson, Ian R; Jones, Nick D; Anderson, Graham; Buckley, Christopher D; Cunningham, Adam F

2014-01-01

The generation of immune cells from BM precursors is a carefully regulated process. This is essential to limit the potential for oncogenesis and autoimmunity yet protect against infection. How infection modulates this is unclear. Salmonella can colonize systemic sites including the BM and spleen. This resolving infection has multiple IFN-γ-mediated acute and chronic effects on BM progenitors, and during the first week of infection IFN-γ is produced by myeloid, NK, NKT, CD4+ T cells, and some lineage-negative cells. After infection, the phenotype of BM progenitors rapidly but reversibly alters, with a peak ∼30-fold increase in Sca-1hi progenitors and a corresponding loss of Sca-1lo/int subsets. Most strikingly, the capacity of donor Sca-1hi cells to reconstitute an irradiated host is reduced; the longer donor mice are exposed to infection, and Sca-1hic-kitint cells have an increased potential to generate B1a-like cells. Thus, Salmonella can have a prolonged influence on BM progenitor functionality not directly related to bacterial persistence. These results reflect changes observed in leucopoiesis during aging and suggest that BM functionality can be modulated by life-long, periodic exposure to infection. Better understanding of this process could offer novel therapeutic opportunities to modulate BM functionality and promote healthy aging. PMID:24825601

Active tissue stiffness modulation controls valve interstitial cell phenotype and osteogenic potential in 3D culture.

PubMed

Duan, Bin; Yin, Ziying; Hockaday Kang, Laura; Magin, Richard L; Butcher, Jonathan T

2016-05-01

Calcific aortic valve disease (CAVD) progression is a highly dynamic process whereby normally fibroblastic valve interstitial cells (VIC) undergo osteogenic differentiation, maladaptive extracellular matrix (ECM) composition, structural remodeling, and tissue matrix stiffening. However, how VIC with different phenotypes dynamically affect matrix properties and how the altered matrix further affects VIC phenotypes in response to physiological and pathological conditions have not yet been determined. In this study, we develop 3D hydrogels with tunable matrix stiffness to investigate the dynamic interplay between VIC phenotypes and matrix biomechanics. We find that VIC populated within hydrogels with valve leaflet like stiffness differentiate towards myofibroblasts in osteogenic media, but surprisingly undergo osteogenic differentiation when cultured within lower initial stiffness hydrogels. VIC differentiation progressively stiffens the hydrogel microenvironment, which further upregulates both early and late osteogenic markers. These findings identify a dynamic positive feedback loop that governs acceleration of VIC calcification. Temporal stiffening of pathologically lower stiffness matrix back to normal level, or blocking the mechanosensitive RhoA/ROCK signaling pathway, delays the osteogenic differentiation process. Therefore, direct ECM biomechanical modulation can affect VIC phenotypes towards and against osteogenic differentiation in 3D culture. These findings highlight the importance of the homeostatic maintenance of matrix stiffness to restrict pathological VIC differentiation. We implement 3D hydrogels with tunable matrix stiffness to investigate the dynamic interaction between valve interstitial cells (VIC, major cell population in heart valve) and matrix biomechanics. This work focuses on how human VIC responses to changing 3D culture environments. Our findings identify a dynamic positive feedback loop that governs acceleration of VIC calcification, which is the hallmark of calcific aortic valve disease. Temporal stiffening of pathologically lower stiffness matrix back to normal level, or blocking the mechanosensitive signaling pathway, delays VIC osteogenic differentiation. Our findings provide an improved understanding of VIC-matrix interactions to aid in interpretation of VIC calcification studies in vitro and suggest that ECM disruption resulting in local tissue stiffness decreases may promote calcific aortic valve disease. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Phenotype heterogeneity in cancer cell populations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Almeida, Luis; Chisholm, Rebecca; Clairambault, Jean

2016-06-08

Phenotype heterogeneity in cancer cell populations, be it of genetic, epigenetic or stochastic origin, has been identified as a main source of resistance to drug treatments and a major source of therapeutic failures in cancers. The molecular mechanisms of drug resistance are partly understood at the single cell level (e.g., overexpression of ABC transporters or of detoxication enzymes), but poorly predictable in tumours, where they are hypothesised to rely on heterogeneity at the cell population scale, which is thus the right level to describe cancer growth and optimise its control by therapeutic strategies in the clinic. We review a fewmore » results from the biological literature on the subject, and from mathematical models that have been published to predict and control evolution towards drug resistance in cancer cell populations. We propose, based on the latter, optimisation strategies of combined treatments to limit emergence of drug resistance to cytotoxic drugs in cancer cell populations, in the monoclonal situation, which limited as it is still retains consistent features of cell population heterogeneity. The polyclonal situation, that may be understood as “bet hedging” of the tumour, thus protecting itself from different sources of drug insults, may lie beyond such strategies and will need further developments. In the monoclonal situation, we have designed an optimised therapeutic strategy relying on a scheduled combination of cytotoxic and cytostatic treatments that can be adapted to different situations of cancer treatments. Finally, we review arguments for biological theoretical frameworks proposed at different time and development scales, the so-called atavistic model (diachronic view relying on Darwinian genotype selection in the coursof billions of years) and the Waddington-like epigenetic landscape endowed with evolutionary quasi-potential (synchronic view relying on Lamarckian phenotype instruction of a given genome by reversible mechanisms), to represent evolution towards heterogeneity, possibly polyclonal, in cancer cell populations and propose innovative directions for therapeutic strategies based on such frameworks.« less

Phenotype heterogeneity in cancer cell populations

NASA Astrophysics Data System (ADS)

Almeida, Luis; Chisholm, Rebecca; Clairambault, Jean; Escargueil, Alexandre; Lorenzi, Tommaso; Lorz, Alexander; Trélat, Emmanuel

2016-06-01

Phenotype heterogeneity in cancer cell populations, be it of genetic, epigenetic or stochastic origin, has been identified as a main source of resistance to drug treatments and a major source of therapeutic failures in cancers. The molecular mechanisms of drug resistance are partly understood at the single cell level (e.g., overexpression of ABC transporters or of detoxication enzymes), but poorly predictable in tumours, where they are hypothesised to rely on heterogeneity at the cell population scale, which is thus the right level to describe cancer growth and optimise its control by therapeutic strategies in the clinic. We review a few results from the biological literature on the subject, and from mathematical models that have been published to predict and control evolution towards drug resistance in cancer cell populations. We propose, based on the latter, optimisation strategies of combined treatments to limit emergence of drug resistance to cytotoxic drugs in cancer cell populations, in the monoclonal situation, which limited as it is still retains consistent features of cell population heterogeneity. The polyclonal situation, that may be understood as "bet hedging" of the tumour, thus protecting itself from different sources of drug insults, may lie beyond such strategies and will need further developments. In the monoclonal situation, we have designed an optimised therapeutic strategy relying on a scheduled combination of cytotoxic and cytostatic treatments that can be adapted to different situations of cancer treatments. Finally, we review arguments for biological theoretical frameworks proposed at different time and development scales, the so-called atavistic model (diachronic view relying on Darwinian genotype selection in the coursof billions of years) and the Waddington-like epigenetic landscape endowed with evolutionary quasi-potential (synchronic view relying on Lamarckian phenotype instruction of a given genome by reversible mechanisms), to represent evolution towards heterogeneity, possibly polyclonal, in cancer cell populations and propose innovative directions for therapeutic strategies based on such frameworks.

Horizontal Transmission of Cytosolic Sup35 Prions by Extracellular Vesicles.

PubMed

Liu, Shu; Hossinger, André; Hofmann, Julia P; Denner, Philip; Vorberg, Ina M

2016-07-12

Prions are infectious protein particles that replicate by templating their aggregated state onto soluble protein of the same type. Originally identified as the causative agent of transmissible spongiform encephalopathies, prions in yeast (Saccharomyces cerevisiae) are epigenetic elements of inheritance that induce phenotypic changes of their host cells. The prototype yeast prion is the translation termination factor Sup35. Prions composed of Sup35 or its modular prion domain NM are heritable and are transmitted vertically to progeny or horizontally during mating. Interestingly, in mammalian cells, protein aggregates derived from yeast Sup35 NM behave as true infectious entities that employ dissemination strategies similar to those of mammalian prions. While transmission is most efficient when cells are in direct contact, we demonstrate here that cytosolic Sup35 NM prions are also released into the extracellular space in association with nanometer-sized membrane vesicles. Importantly, extracellular vesicles are biologically active and are taken up by recipient cells, where they induce self-sustained Sup35 NM protein aggregation. Thus, in mammalian cells, extracellular vesicles can serve as dissemination vehicles for protein-based epigenetic information transfer. Prions are proteinaceous infectious particles that propagate by templating their quaternary structure onto nascent proteins of the same kind. Prions in yeast act as heritable epigenetic elements that can alter the phenotype when transmitted to daughter cells or during mating. Prion activity is conferred by so-called prion domains often enriched in glutamine and asparagine residues. Interestingly, many mammalian proteins also contain domains with compositional similarity to yeast prion domains. We have recently provided a proof-of-principle demonstration that a yeast prion domain also retains its prion activity in mammalian cells. We demonstrate here that cytosolic prions composed of a yeast prion domain are also packaged into extracellular vesicles that transmit the prion phenotype to bystander cells. Thus, proteins with prion-like domains can behave as proteinaceous information molecules that exploit the cellular vesicle trafficking machinery for intercellular long-distance dissemination. Copyright © 2016 Liu et al.

Semaphorin 4D in human head and neck cancer tissue and peripheral blood: A dense fibrotic peri-tumoral stromal phenotype

PubMed Central

Derakhshandeh, Roshanak; Sanadhya, Sonia; Lee Han, Kyu; Chen, Haiyan; Goloubeva, Olga; Webb, Tonya J.; Younis, Rania H.

2018-01-01

The search for stromal biomarkers in carcinoma patients is a challenge in the field. Semaphorin 4D (Sema4D), known for its various developmental, physiological and pathological effects, plays a role in pro and anti-inflammatory responses. It is expressed in many epithelial tumors including head and neck squamous cell carcinoma (HNSCC). Recently, we found that HNSCC-associated Sema4D modulates an immune-suppressive, tumor-permissible environment by inducing the expansion of myeloid derived suppressor cells. The purpose of this study was to determine the value of Sema4D as a biomarker for the peri-tumoral stromal phenotype in human HNSCC. Our data showed Sema4D+ve/high tumor cells in 34% of the studied cohort with positive correlation to Stage III (p=0.0001). Sema4D+ve/high tumor cells correlated directly with dense fibrotic peri-tumoral stroma (p=0.0001) and inversely with infiltrate of Sema4D+ve/high tumor-associated inflammatory cells (TAIs) (p=0.01). Most of the Sema4D+ve/high TAIs were co-positive for the macrophage biomarker CD163. Knockdown of Sema4D in WSU-HN6 cells inhibited collagen production by fibroblasts, and decreased activated TGF-β1 levels in culture medium of HNSCC cell lines. In a stratification model of HNSCC using combined Sema4D and the programmed death ligand 1 (PDL-1), Sema4D+ve/high tumor cells represented a phenotype distinct from the PDL-1 positive tumors. Finally,Sema4D was detected in plasma of HNC patients at significantly higher levels (115.44, ± 39.37) compared to healthy donors (38.60± 12.73) (p <0.0001). In conclusion, we present a novel HNSCC tumor stratification model, based on the expression of the biomarker Sema4D. This model opens new avenues to novel targeted therapeutic strategies. PMID:29541402

Human and feline adipose-derived mesenchymal stem cells have comparable phenotype, immunomodulatory functions, and transcriptome.

PubMed

Clark, Kaitlin C; Fierro, Fernando A; Ko, Emily Mills; Walker, Naomi J; Arzi, Boaz; Tepper, Clifford G; Dahlenburg, Heather; Cicchetto, Andrew; Kol, Amir; Marsh, Lyndsey; Murphy, William J; Fazel, Nasim; Borjesson, Dori L

2017-03-20

Adipose-derived mesenchymal stem cells (ASCs) are a promising cell therapy to treat inflammatory and immune-mediated diseases. Development of appropriate pre-clinical animal models is critical to determine safety and attain early efficacy data for the most promising therapeutic candidates. Naturally occurring diseases in cats already serve as valuable models to inform human clinical trials in oncologic, cardiovascular, and genetic diseases. The objective of this study was to complete a comprehensive side-by-side comparison of human and feline ASCs, with an emphasis on their immunomodulatory capacity and transcriptome. Human and feline ASCs were evaluated for phenotype, immunomodulatory profile, and transcriptome. Additionally, transwells were used to determine the role of cell-cell contact in ASC-mediated inhibition of lymphocyte proliferation in both humans and cats. Similar to human ASCs, feline ASCs were highly proliferative at low passages and fit the minimal criteria of multipotent stem cells including a compatible surface protein phenotype, osteogenic capacity, and normal karyotype. Like ASCs from all species, feline ASCs inhibited mitogen-activated lymphocyte proliferation in vitro, with or without direct ASC-lymphocyte contact. Feline ASCs mimic human ASCs in their mediator secretion pattern, including prostaglandin E2, indoleamine 2,3 dioxygenase, transforming growth factor beta, and interleukin-6, all augmented by interferon gamma secretion by lymphocytes. The transcriptome of three unactivated feline ASC lines were highly similar. Functional analysis of the most highly expressed genes highlighted processes including: 1) the regulation of apoptosis; 2) cell adhesion; 3) response to oxidative stress; and 4) regulation of cell differentiation. Finally, feline ASCs had a similar gene expression profile to noninduced human ASCs. Findings suggest that feline ASCs modulate lymphocyte proliferation using soluble mediators that mirror the human ASC secretion pattern. Uninduced feline ASCs have similar gene expression profiles to uninduced human ASCs, as revealed by transcriptome analysis. These data will help inform clinical trials using cats with naturally occurring diseases as surrogate models for human clinical trials in the regenerative medicine arena.

Regulatory T Cells Subvert Mycobacterial Containment in Patients Failing Extensively Drug-resistant TB Treatment.

PubMed

Davids, Malika; Pooran, Anil S; Pietersen, Elize; Wainwright, Helen C; Binder, Anke; Warren, Robin; Dheda, Keertan

2018-02-09

The advent of extensively (XDR-TB) and totally drug-resistant TB, with limited or no treatment options, has facilitated renewed interest in host directed immunotherapy, particularly for therapeutically destitute patients. However, the selection and utility of such approaches depend upon understanding the host immune response in XDR-TB, which hitherto remains unexplored. To determine the host immunological profile in patients with XDR-TB, compared to drug-sensitive TB, using peripheral blood and explanted lung tissue. Blood and explanted lung tissue were obtained from patients with XDR-TB (n=31), drug-sensitive TB (DS-TB, n=20) and presumed latent-TB infection (LTBI, n=20). T-cell phenotype (Th1/Th2/Th17/Tregs) was evaluated in all patient groups, and Treg function assessed in XDR-TB non-responders by co-culturing PPD pre-primed effector T-cells with H37Rv-infected monocyte-derived macrophages, with or without autologous Tregs. Mycobacterial containment was evaluated by counting colony-forming units. Patients failing XDR-TB treatment had an altered immuno-phenotype characterized by a substantial increase in the frequency (median; IQR) of CD4+CD25+FoxP3+ regulatory T-cells (11.5; 5.9-15.2) compared to DS-TB (3.4 %; 1.6-5.73; p < 0.001) and presumed LTBI (1.8 % 1.2-2.3; p < 0.001), which was unrelated to disease duration. Tregs isolated from XDR-TB patients suppressed T-cell proliferation (up to 90%) and subverted containment of H37Rv-infected monocyte-derived macrophages (by 30%; p= 0.03) by impairing effector T-cell function through a mechanism independent of direct cell-to-cell contact, IL-10, TGF-beta and CTLA-4. Collectively, these data suggest that Tregs may be contributing to immune dysfunction, and bacterial persistence, in patients with XDR-TB. The relevant cellular pathways may serve as potential targets for immunotherapeutic intervention.

Generational comparisons (F1 versus F3) of vinclozolin induced epigenetic transgenerational inheritance of sperm differential DNA methylation regions (epimutations) using MeDIP-Seq.

PubMed

Beck, Daniel; Sadler-Riggleman, Ingrid; Skinner, Michael K

2017-07-01

Environmentally induced epigenetic transgenerational inheritance of disease and phenotypic variation has been shown to involve DNA methylation alterations in the germline (e.g. sperm). These differential DNA methylation regions (DMRs) are termed epimutations and in part transmit the transgenerational phenotypes. The agricultural fungicide vinclozolin exposure of a gestating female rat has previously been shown to promote transgenerational disease and epimutations in F3 generation (great-grand-offspring) animals. The current study was designed to investigate the actions of direct fetal exposure on the F1 generation rat sperm DMRs compared to the F3 transgenerational sperm DMRs. A protocol involving methylated DNA immunoprecipitation (MeDIP) followed by next-generation sequencing (Seq) was used in the current study. Bioinformatics analysis of the MeDIP-Seq data was developed and several different variations in the bioinformatic analysis were evaluated. Observations indicate needs to be considered. Interestingly, the F1 generation DMRs were found to be fewer in number and for the most part distinct from the F3 generation epimutations. Observations suggest the direct exposure induced F1 generation sperm DMRs appear to promote in subsequent generations alterations in the germ cell developmental programming that leads to the distinct epimutations in the F3 generation. This may help explain the differences in disease and phenotypes between the direct exposure F1 generation and transgenerational F3 generation. Observations demonstrate a distinction between the direct exposure versus transgenerational epigenetic programming induced by environmental exposures and provide insights into the molecular mechanisms involved in the epigenetic transgenerational inheritance phenomenon.

Nasal lavage, blood or sputum: Which is best for phenotyping asthma?

PubMed

de Farias, Camyla F; Amorim, Maria M F; Dracoulakis, Michel; Caetano, Lilian B; Santoro, Ilka L; Fernandes, Ana L G

2017-05-01

Determination of asthma phenotypes, particularly inflammatory phenotypes, helps guide treatment and management of this heterogeneous disease. Induced sputum cytology has been the gold standard for determination of inflammatory phenotypes, but sputum induction is fairly invasive and technically challenging. Blood and nasal lavage cytology have been suggested as substitutes, but have not been fully verified. The aim of this study is to determine the accuracy of blood and nasal lavage cytometry as indicators of inflammatory phenotypes in asthma. Clinical evaluation, Asthma Control Questionnaire (ACQ) and spirometry were performed for 121 adult asthma patients, and blood, nasal lavage and induced sputum samples were taken. Eosinophils and neutrophils were counted in three samples from each subject. Inflammatory phenotypes (eosinophilic, neutrophilic, mixed and paucicellular) and cells counts were analysed using Venn diagram and receiver operating characteristic (ROC) curve, respectively. ACQ score, spirometry and bronchodilator response did not differ among subjects with different inflammatory phenotypes. Inflammatory phenotypes defined by nasal lavage cytometry were in better concordance than those defined by blood cell counts with phenotypes determined by sputum cytology, and were significantly correlated with sputum phenotypes. For eosinophilia, nasal lavage cytology showed better accuracy than blood cytology (area under the curve (AUC): 0.89 vs 0.65). For all phenotypes, sensitivity and positive and negative predictive power were higher for nasal lavage cytometry than for blood. Blood cell counts gave a high level of false positives for all inflammatory phenotypes. We recommend nasal lavage cytology over blood cell count as a substitute for sputum cytology to identify inflammatory phenotypes in asthma. © 2016 Asian Pacific Society of Respirology.

Antitumor killer lymphocytes in the peripheral blood of a patient with transitional cell carcinoma of the bladder.

PubMed

Kim, C J; Yuasa, T; Kushima, R; Tomoyoshi, T; Seto, A

1998-05-01

Peripheral blood lymphocytes (PBL) from patients with bladder cancer also contain cells possessing cytotoxic activity against autologous tumor cells. These cells are phenotypically heterogenous and include natural killer (NK) and cytotoxic T cells. This study investigated the role of cytotoxic lymphocytes directed against autologous bladder cancer cells. PBL were obtained at intervals before and after surgery and analyzed for cytotoxic activity against autologous bladder cancer cells in 4-hour 51Cr release assay. PBL stimulated with autologous tumor cells were also transformed with human T-lymphotropic virus type-1, establishing a cell line (KB31) which was analyzed for phenotype and cytotoxic activity against the autologous tumor cells. PBL preoperative cytotoxic activity was low, but increased after surgery. Cytotoxic activity was found not only against autologous bladder cancer cells, but also against heterologous bladder cancer (KK-47) and myeloid leukemia (K562) cells, with the highest activity against the heterologous cell lines. The cytotoxic activity of KB31 was 40% against autologous tumor cells 6 weeks after initiation of the cell line, but decreased to 5% by 6 months. This activity was lower than that against the other cell lines, and was similar to that of PBL in short-term culture. Fluorescence-activated cell sorter (FACS) analysis demonstrated that in KB31 cells at 6 weeks, CD8+ cells were dominant, but CD56+ cells predominated at 6 months. These results suggest that the presence of cytotoxic activity in the peripheral blood of the patient was due to both cytotoxic T cells and NK cells. The cytotoxic activity was lowest prior to surgery and increased postoperatively.

Upregulation of miR-3607 promotes lung adenocarcinoma proliferation by suppressing APC expression.

PubMed

Lin, Yong; Gu, Qiangye; Sun, Zongwen; Sheng, Baowei; Qi, Congcong; Liu, Bing; Fu, Tian; Liu, Cun; Zhang, Yan

2017-11-01

Lung cancer is the leading cause of worldwide cancer-related deaths, although many drugs and new therapeutic approaches have been used, the 5-years survival rate is still low for lung cancer patients. microRNAs have been shown to regulate lung cancer initiation and development, here we studied the role of miR-3607 in lung cancer cell proliferation. We found miR-3607 was upregulated in lung cancer tissues and cells, miR-3607 overexpression promoted lung cancer cell A549 proliferation determined by MTT assay, colony formation assay, anchorage-independent growth ability assay and bromodeoxyuridine incorporation assay, while the opposite phenotypes were shown when miR-3607 was knocked down. Predicted analysis suggested a Wnt signaling pathway regulator adenomatous polyposis coli (APC) was the target of miR-3607, miR-3607 could directly bind to the 3'UTR of APC, and promoted Cyclin D1 and c-Myc expression which can be suppressed by APC. Double knockdown of miR-3607 and APC copied the phenotypes of miR-3607 overexpression, suggesting miR-3607 promoted lung cancer cell A549 proliferation by targeting APC. In conclusion, our study suggested miR-3607 contributes to lung cancer cell proliferation by inhibiting APC. Copyright © 2017. Published by Elsevier Masson SAS.

Senescent intervertebral disc cells exhibit perturbed matrix homeostasis phenotype.

PubMed

Ngo, Kevin; Patil, Prashanti; McGowan, Sara J; Niedernhofer, Laura J; Robbins, Paul D; Kang, James; Sowa, Gwendolyn; Vo, Nam

2017-09-01

Aging greatly increases the risk for intervertebral disc degeneration (IDD) as a result of proteoglycan loss due to reduced synthesis and enhanced degradation of the disc matrix proteoglycan (PG). How disc matrix PG homeostasis becomes perturbed with age is not known. The goal of this study is to determine whether cellular senescence is a source of this perturbation. We demonstrated that disc cellular senescence is dramatically increased in the DNA repair-deficient Ercc1 -/Δ mouse model of human progeria. In these accelerated aging mice, increased disc cellular senescence is closely associated with the rapid loss of disc PG. We also directly examine PG homeostasis in oxidative damage-induced senescent human cells using an in vitro cell culture model system. Senescence of human disc cells treated with hydrogen peroxide was confirmed by growth arrest, senescence-associated β-galactosidase activity, γH2AX foci, and acquisition of senescence-associated secretory phenotype. Senescent human disc cells also exhibited perturbed matrix PG homeostasis as evidenced by their decreased capacity to synthesize new matrix PG and enhanced degradation of aggrecan, a major matrix PG. of the disc. Our in vivo and in vitro findings altogether suggest that disc cellular senescence is an important driver of PG matrix homeostatic perturbation and PG loss. Published by Elsevier B.V.

Characterization of colonic dendritic cells in normal and colitic mice.

PubMed

Cruickshank, Sheena M; English, Nicholas R; Felsburg, Peter J; Carding, Simon R

2005-10-28

Recent studies demonstrating the direct involvement of dendritic cells (DC) in the activation of pathogenic T cells in animal models of inflammatory bowel disease identify DC as important antigen presenting cells in the colon. However, very little is known about the properties of colonic DC. Using immunohistochemistry, electron microscopy and flow cytometry we have characterized and compared colonic DC in the colon of healthy animals and interleukin-2-deficient (IL2(-/-)) mice that develop colitis. In the healthy colon, DC resided within the lamina propria and in close association with the basement membrane of colonic villi. Type 1 myeloid (CD11c(+), CD11b(+), B220(-), CD8alpha(-)) DC made up the largest (40-45%) population and all DC expressed low levels of CD80, CD86, and CD40, and had high endocytic activity consistent with an immature phenotype. In colitic IL2(-/-) mice, colonic DC numbers increased four- to five-fold and were localized within the epithelial layer and within aggregates of T and B cells. They were also many more DC in mesenteric lymph nodes (MLN). The majority (>85%) of DC in the colon and MLN of IL2(-/-) mice were type 1 myeloid, and expressed high levels of MHC class II, CD80, CD86, CD40, DEC 205, and CCR5 molecules and were of low endocytic activity consistent with mature DC. These findings demonstrate striking changes in the number, distribution and phenotype of DC in the inflamed colon. Their intimate association with lymphocytes in the colon and draining lymph nodes suggest that they may contribute directly to the ongoing inflammation in the colon.

Characterization of colonic dendritic cells in normal and colitic mice

PubMed Central

Cruickshank, Sheena M; English, Nicholas R; Felsburg, Peter J; Carding, Simon R

2005-01-01

AIM: Recent studies demonstrating the direct involvement of dendritic cells (DC) in the activation of pathogenic T cells in animal models of inflammatory bowel disease identify DC as important antigen presenting cells in the colon. However, very little is known about the properties of colonic DC. METHODS: Using immunohistochemistry, electron microscopy and flow cytometry we have characterized and compared colonic DC in the colon of healthy animals and interleukin-2-deficient (IL2-/-) mice that develop colitis. RESULTS: In the healthy colon, DC resided within the lamina propria and in close association with the basement membrane of colonic villi. Type 1 myeloid (CD11c+, CD11b+, B220-, CD8α-) DC made up the largest (40-45%) population and all DC expressed low levels of CD80, CD86, and CD40, and had high endocytic activity consistent with an immature phenotype. In colitic IL2-/- mice, colonic DC numbers increased four- to five-fold and were localized within the epithelial layer and within aggregates of T and B cells. They were also many more DC in mesenteric lymph nodes (MLN). The majority (>85%) of DC in the colon and MLN of IL2-/- mice were type 1 myeloid, and expressed high levels of MHC class II, CD80, CD86, CD40, DEC 205, and CCR5 molecules and were of low endocytic activity consistent with mature DC. CONCLUSION: These findings demonstrate striking changes in the number, distribution and phenotype of DC in the inflamed colon. Their intimate association with lymphocytes in the colon and draining lymph nodes suggest that they may contribute directly to the ongoing inflammation in the colon. PMID:16419163

Reduced sympathetic innervation after alteration of target cell neurotransmitter phenotype in transgenic mice.

PubMed Central

Cho, S; Son, J H; Park, D H; Aoki, C; Song, X; Smith, G P; Joh, T H

1996-01-01

Neurotransmitters play a variety of important roles during nervous system development. In the present study, we hypothesized that neurotransmitter phenotype of both projecting and target cells is an important factor for the final synaptic linkage and its specificity. To test this hypothesis, we used transgenic techniques to convert serotonin/melatonin-producing cells of the pineal gland into cells that also produce dopamine and investigated the innervation of the phenotypically altered target cells. This phenotypic alteration markedly reduced the noradrenergic innervation originating from the superior cervical ganglia. Although the mechanism by which the reduction occurs is presently unknown, quantitative enzyme-linked immunoassay showed the presence of the equivalent amounts of nerve growth factor (NGF) in the control and transgenic pineal glands, suggesting that it occurred in a NGF-independent manner. The results suggest that target neurotransmitter phenotype influences the formation of afferent connections during development. Images Fig. 3 Fig. 4 PMID:8610132

Generation and characterization of p53 null transformed hepatic progenitor cells: oval cells give rise to hepatocellular carcinoma.

PubMed

Dumble, Melissa L; Croager, Emma J; Yeoh, George C T; Quail, Elizabeth A

2002-03-01

Oval cells are bipotential liver stem cells able to differentiate into hepatocytes and bile duct epithelia. In normal adult liver oval cells are quiescent, existing in low numbers around the periportal region, and proliferate following severe, prolonged liver trauma. There is evidence implicating oval cells in the development of hepatocellular carcinoma, and hence the availability of an immortalized oval cell line would be invaluable for the study of liver cell lineage differentiation and carcinogenesis. A novel approach in the generation of cell lines is the use of the p53 knockout mouse. Absence of p53 allows a cell to cycle past the normal Hayflick limit, rendering it immortalized, although subsequent genetic alterations are thought necessary for transformation. p53 knockout mice were fed a choline-deficient, ethionine-supplemented diet, previously shown to increase oval cell numbers in wild-type mice. The oval cells were isolated by centrifugal elutriation and maintained in culture. Colonies of hepatic cells were isolated and characterized with respect to phenotype, growth characteristics and tumorigenicity. Analysis of gene expression by Northern blotting and immunocytochemistry suggests they are oval-like cells by virtue of albumin and transferrin expression, as well as the oval cell markers alpha fetoprotein, M(2)-pyruvate kinase and A6. Injection into athymic nude mice shows the cell lines are capable of forming tumors which phenotypically resemble hepatocellular carcinoma. Thus, the use of p53 null hepatic cells successfully generated immortalized and tumorigenic hepatic stem cell lines. The results presented support the idea that deleting p53 allows immortalization and contributes to the transformation of the oval-like cell lines. Further, the tumorigenic status of the cell lines is direct evidence for the participation of oval cells in the formation of hepatocellular carcinoma.

Hepatic Oval Cells Have the Side Population Phenotype Defined by Expression of ATP-Binding Cassette Transporter ABCG2/BCRP1

PubMed Central

Shimano, Koichi; Satake, Makoto; Okaya, Atsuhito; Kitanaka, Junichi; Kitanaka, Nobue; Takemura, Motohiko; Sakagami, Masafumi; Terada, Nobuyuki; Tsujimura, Tohru

2003-01-01

Organ-specific stem cells can be identified by the side population (SP) phenotype, which is defined by the property to effectively exclude the Hoechst 33342 dye. The ATP-binding cassette transporter ABCG2/BCRP1 mediates the SP phenotype. Because hepatic oval cells possess several characteristics of stem cells, we examined whether they have the SP phenotype using the 2-acetylaminofluorene/partial hepatectomy (PH) model. Fluorescence-activated cell sorting analysis showed that a population of non-parenchymal cells containing oval cells, prepared on day 7 after PH, carried a significant number of SP cells, whereas that of non-parenchymal cells without oval cells, prepared on day 0 after PH, did not. Northern blot analysis using total liver RNA obtained on various days after PH showed that the expression of ABCG2/BCRP1 mRNA increased after PH, reaching the highest level on day 7, and then gradually decreased. This pattern of changes in the ABCG2/BCRP1 mRNA level was well correlated to that in the number of oval cells. Furthermore, in situ hybridization revealed that oval cells were the sites of expression of ABCG2/BCRP1 mRNA. These results indicate that oval cells have the SP phenotype defined by expression of ABCG2/BCRP1, suggesting that oval cells may represent stem cells in the liver. PMID:12819005

Over-expression of FoxM1 leads to epithelial-mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells

PubMed Central

Bao, Bin; Wang, Zhiwei; Ali, Shadan; Kong, Dejuan; Banerjee, Sanjeev; Ahmad, Aamir; Li, Yiwei; Azmi, Asfar S.; Miele, Lucio; Sarkar, Fazlul H.

2011-01-01

FoxM1 is known to play important role in the development and progression of many malignancies including pancreatic cancer. Studies have shown that the acquisition of Epithelial-to-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotypes are highly inter-related, and contributes to drug resistance, tumor recurrence and metastasis. The molecular mechanism(s) by which FoxM1 contributes to the acquisition of EMT phenotype and induction of CSC self-renewal capacity is poorly understood. Therefore, we established FoxM1 over-expressing pancreatic cancer (AsPC-1) cells, which showed increased cell growth, clonogenicity and cell migration. Moreover, over-expression of FoxM1 led to the acquisition of EMT phenotype by activation of mesenchymal cell markers, ZEB1, ZEB2, Snail2, E-cadherin, and vimentin, which is consistent with increased sphere-forming (pancreatospheres) capacity and expression of CSC surface markers (CD44 and EpCAM). We also found that over-expression of FoxM1 led to decreased expression of miRNAs (let-7a, let-7b, let-7c, miR-200b and miR-200c); however, re-expression of miR-200b inhibited the expression of ZEB1, ZEB2, vimentin as well as FoxM1, and induced the expression of E-cadherin, leading to the reversal of EMT phenotype. Finally, we found that genistein, a natural chemo-preventive agent, inhibited cell growth, clonogenicity, cell migration and invasion, EMT phenotype, and formation of pancreatospheres consistent with reduced expression of CD44 and EpCAM. These results suggest, for the first time, that FoxM1 over-expression is responsible for the acquisition of EMT and CSC phenotype, which is in part mediated through the regulation of miR-200b and these processes, could be easily attenuated by genistein. PMID:21503965

Marmoset induced pluripotent stem cells: Robust neural differentiation following pretreatment with dimethyl sulfoxide.

PubMed

Qiu, Zhifang; Mishra, Anuja; Li, Miao; Farnsworth, Steven L; Guerra, Bernadette; Lanford, Robert E; Hornsby, Peter J

2015-07-01

The marmoset is an important nonhuman primate model for regenerative medicine. For experimental autologous cell therapy based on induced pluripotent (iPS) cells in the marmoset, cells must be able to undergo robust and reliable directed differentiation that will not require customization for each specific iPS cell clone. When marmoset iPS cells were aggregated in a hanging drop format for 3 days, followed by exposure to dual SMAD inhibitors and retinoic acid in monolayer culture for 3 days, we found substantial variability in the response of different iPS cell clones. However, when clones were pretreated with 0.05-2% dimethyl sulfoxide (DMSO) for 24 hours, all clones showed a very similar maximal response to the directed differentiation scheme. Peak responses were observed at 0.5% DMSO in two clones and at 1% DMSO in a third clone. When patterns of gene expression were examined by microarray analysis, hierarchical clustering showed very similar responses in all 3 clones when they were pretreated with optimal DMSO concentrations. The change in phenotype following exposure to DMSO and the 6 day hanging drop/monolayer treatment was confirmed by immunocytochemistry. Analysis of DNA content in DMSO-exposed cells indicated that it is unlikely that DMSO acts by causing cells to exit from the cell cycle. This approach should be generally valuable in the directed neural differentiation of pluripotent cells for experimental cell therapy. Copyright © 2015. Published by Elsevier B.V.

Developmental changes in electrophysiological characteristics of human-induced pluripotent stem cell-derived cardiomyocytes.

PubMed

Ben-Ari, Meital; Naor, Shulamit; Zeevi-Levin, Naama; Schick, Revital; Ben Jehuda, Ronen; Reiter, Irina; Raveh, Amit; Grijnevitch, Inna; Barak, Omri; Rosen, Michael R; Weissman, Amir; Binah, Ofer

2016-12-01

Previous studies proposed that throughout differentiation of human induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs), only 3 types of action potentials (APs) exist: nodal-, atrial-, and ventricular-like. To investigate whether there are precisely 3 phenotypes or a continuum exists among them, we tested 2 hypotheses: (1) During culture development a cardiac precursor cell is present that-depending on age-can evolve into the 3 phenotypes. (2) The predominant pattern is early prevalence of a nodal phenotype, transient appearance of an atrial phenotype, evolution to a ventricular phenotype, and persistence of transitional phenotypes. To test these hypotheses, we (1) performed fluorescence-activated cell sorting analysis of nodal, atrial, and ventricular markers; (2) recorded APs from 280 7- to 95-day-old iPSC-CMs; and (3) analyzed AP characteristics. The major findings were as follows: (1) fluorescence-activated cell sorting analysis of 30- and 60-day-old cultures showed that an iPSC-CMs population shifts from the nodal to the atrial/ventricular phenotype while including significant transitional populations; (2) the AP population did not consist of 3 phenotypes; (3) culture aging was associated with a shift from nodal to ventricular dominance, with a transient (57-70 days) appearance of the atrial phenotype; and (4) beat rate variability was more prominent in nodal than in ventricular cardiomyocytes, while pacemaker current density increased in older cultures. From the onset of development in culture, the iPSC-CMs population includes nodal, atrial, and ventricular APs and a broad spectrum of transitional phenotypes. The most readily distinguishable phenotype is atrial, which appears only transiently yet dominates at 57-70 days of evolution. Copyright © 2016 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Anti-miR-21 Suppresses Hepatocellular Carcinoma Growth via Broad Transcriptional Network Deregulation.

PubMed

Wagenaar, Timothy R; Zabludoff, Sonya; Ahn, Sung-Min; Allerson, Charles; Arlt, Heike; Baffa, Raffaele; Cao, Hui; Davis, Scott; Garcia-Echeverria, Carlos; Gaur, Rajula; Huang, Shih-Min A; Jiang, Lan; Kim, Deokhoon; Metz-Weidmann, Christiane; Pavlicek, Adam; Pollard, Jack; Reeves, Jason; Rocnik, Jennifer L; Scheidler, Sabine; Shi, Chaomei; Sun, Fangxian; Tolstykh, Tatiana; Weber, William; Winter, Christopher; Yu, Eunsil; Yu, Qunyan; Zheng, Gang; Wiederschain, Dmitri

2015-06-01

Hepatocellular carcinoma (HCC) remains a significant clinical challenge with few therapeutic options available to cancer patients. MicroRNA 21-5p (miR-21) has been shown to be upregulated in HCC, but the contribution of this oncomiR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific single-stranded oligonucleotide inhibitors of miR-21 (anti-miRNAs) and used them to interrogate dependency on miR-21 in a panel of liver cancer cell lines. Treatment with anti-miR-21, but not with a mismatch control anti-miRNA, resulted in significant derepression of direct targets of miR-21 and led to loss of viability in the majority of HCC cell lines tested. Robust induction of caspase activity, apoptosis, and necrosis was noted in anti-miR-21-treated HCC cells. Furthermore, ablation of miR-21 activity resulted in inhibition of HCC cell migration and suppression of clonogenic growth. To better understand the consequences of miR-21 suppression, global gene expression profiling was performed on anti-miR-21-treated liver cancer cells, which revealed striking enrichment in miR-21 target genes and deregulation of multiple growth-promoting pathways. Finally, in vivo dependency on miR-21 was observed in two separate HCC tumor xenograft models. In summary, these data establish a clear role for miR-21 in the maintenance of tumorigenic phenotype in HCC in vitro and in vivo. miR-21 is important for the maintenance of the tumorigenic phenotype of HCC and represents a target for pharmacologic intervention. ©2015 American Association for Cancer Research.

WVD2 and WDL1 modulate helical organ growth and anisotropic cell expansion in Arabidopsis

NASA Technical Reports Server (NTRS)

Yuen, Christen Y L.; Pearlman, Rebecca S.; Silo-Suh, Laura; Hilson, Pierre; Carroll, Kathleen L.; Masson, Patrick H.

2003-01-01

Wild-type Arabidopsis roots develop a wavy pattern of growth on tilted agar surfaces. For many Arabidopsis ecotypes, roots also grow askew on such surfaces, typically slanting to the right of the gravity vector. We identified a mutant, wvd2-1, that displays suppressed root waving and leftward root slanting under these conditions. These phenotypes arise from transcriptional activation of the novel WAVE-DAMPENED2 (WVD2) gene by the cauliflower mosaic virus 35S promoter in mutant plants. Seedlings overexpressing WVD2 exhibit constitutive right-handed helical growth in both roots and etiolated hypocotyls, whereas the petioles of WVD2-overexpressing rosette leaves exhibit left-handed twisting. Moreover, the anisotropic expansion of cells is impaired, resulting in the formation of shorter and stockier organs. In roots, the phenotype is accompanied by a change in the arrangement of cortical microtubules within peripheral cap cells and cells at the basal end of the elongation zone. WVD2 transcripts are detectable by reverse transcriptase-polymerase chain reaction in multiple organs of wild-type plants. Its predicted gene product contains a conserved region named "KLEEK," which is found only in plant proteins. The Arabidopsis genome possesses seven other genes predicted to encode KLEEK-containing products. Overexpression of one of these genes, WVD2-LIKE 1, which encodes a protein with regions of similarity to WVD2 extending beyond the KLEEK domain, results in phenotypes that are highly similar to wvd2-1. Silencing of WVD2 and its paralogs results in enhanced root skewing in the wild-type direction. Our observations suggest that at least two members of this gene family may modulate both rotational polarity and anisotropic cell expansion during organ growth.

Live Imaging at the Onset of Cortical Neurogenesis Reveals Differential Appearance of the Neuronal Phenotype in Apical versus Basal Progenitor Progeny

PubMed Central

Attardo, Alessio; Calegari, Federico; Haubensak, Wulf; Wilsch-Bräuninger, Michaela; Huttner, Wieland B.

2008-01-01

The neurons of the mammalian brain are generated by progenitors dividing either at the apical surface of the ventricular zone (neuroepithelial and radial glial cells, collectively referred to as apical progenitors) or at its basal side (basal progenitors, also called intermediate progenitors). For apical progenitors, the orientation of the cleavage plane relative to their apical-basal axis is thought to be of critical importance for the fate of the daughter cells. For basal progenitors, the relationship between cell polarity, cleavage plane orientation and the fate of daughter cells is unknown. Here, we have investigated these issues at the very onset of cortical neurogenesis. To directly observe the generation of neurons from apical and basal progenitors, we established a novel transgenic mouse line in which membrane GFP is expressed from the beta-III-tubulin promoter, an early pan-neuronal marker, and crossed this line with a previously described knock-in line in which nuclear GFP is expressed from the Tis21 promoter, a pan-neurogenic progenitor marker. Mitotic Tis21-positive basal progenitors nearly always divided symmetrically, generating two neurons, but, in contrast to symmetrically dividing apical progenitors, lacked apical-basal polarity and showed a nearly randomized cleavage plane orientation. Moreover, the appearance of beta-III-tubulin–driven GFP fluorescence in basal progenitor-derived neurons, in contrast to that in apical progenitor-derived neurons, was so rapid that it suggested the initiation of the neuronal phenotype already in the progenitor. Our observations imply that (i) the loss of apical-basal polarity restricts neuronal progenitors to the symmetric mode of cell division, and that (ii) basal progenitors initiate the expression of neuronal phenotype already before mitosis, in contrast to apical progenitors. PMID:18545663

Coordination of planar cell polarity pathways through Spiny-legs

PubMed Central

Ambegaonkar, Abhijit A; Irvine, Kenneth D

2015-01-01

Morphogenesis and physiology of tissues and organs requires planar cell polarity (PCP) systems that orient and coordinate cells and their behaviors, but the relationship between PCP systems has been controversial. We have characterized how the Frizzled and Dachsous-Fat PCP systems are connected through the Spiny-legs isoform of the Prickle-Spiny-legs locus. Two different components of the Dachsous-Fat system, Dachsous and Dachs, can each independently interact with Spiny-legs and direct its localization in vivo. Through characterization of the contributions of Prickle, Spiny-legs, Dachsous, Fat, and Dachs to PCP in the Drosophila wing, eye, and abdomen, we define where Dachs-Spiny-legs and Dachsous-Spiny-legs interactions contribute to PCP, and provide a new understanding of the orientation of polarity and the basis of PCP phenotypes. Our results support the direct linkage of PCP systems through Sple in specific locales, while emphasizing that cells can be subject to and must ultimately resolve distinct, competing PCP signals. DOI: http://dx.doi.org/10.7554/eLife.09946.001 PMID:26505959

Macrophage-Derived Extracellular Succinate Licenses Neural Stem Cells to Suppress Chronic Neuroinflammation.

PubMed

Peruzzotti-Jametti, Luca; Bernstock, Joshua D; Vicario, Nunzio; Costa, Ana S H; Kwok, Chee Keong; Leonardi, Tommaso; Booty, Lee M; Bicci, Iacopo; Balzarotti, Beatrice; Volpe, Giulio; Mallucci, Giulia; Manferrari, Giulia; Donegà, Matteo; Iraci, Nunzio; Braga, Alice; Hallenbeck, John M; Murphy, Michael P; Edenhofer, Frank; Frezza, Christian; Pluchino, Stefano

2018-03-01

Neural stem cell (NSC) transplantation can influence immune responses and suppress inflammation in the CNS. Metabolites, such as succinate, modulate the phenotype and function of immune cells, but whether and how NSCs are also activated by such immunometabolites to control immunoreactivity and inflammatory responses is unclear. Here, we show that transplanted somatic and directly induced NSCs ameliorate chronic CNS inflammation by reducing succinate levels in the cerebrospinal fluid, thereby decreasing mononuclear phagocyte (MP) infiltration and secondary CNS damage. Inflammatory MPs release succinate, which activates succinate receptor 1 (SUCNR1)/GPR91 on NSCs, leading them to secrete prostaglandin E2 and scavenge extracellular succinate with consequential anti-inflammatory effects. Thus, our work reveals an unexpected role for the succinate-SUCNR1 axis in somatic and directly induced NSCs, which controls the response of stem cells to inflammatory metabolic signals released by type 1 MPs in the chronically inflamed brain. Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.

Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides

PubMed Central

Wefers, Benedikt; Meyer, Melanie; Ortiz, Oskar; Hrabé de Angelis, Martin; Hansen, Jens; Wurst, Wolfgang; Kühn, Ralf

2013-01-01

The study of genetic disease mechanisms relies mostly on targeted mouse mutants that are derived from engineered embryonic stem (ES) cells. Nevertheless, the establishment of mutant ES cells is laborious and time-consuming, restricting the study of the increasing number of human disease mutations discovered by high-throughput genomic analysis. Here, we present an advanced approach for the production of mouse disease models by microinjection of transcription activator-like effector nucleases (TALENs) and synthetic oligodeoxynucleotides into one-cell embryos. Within 2 d of embryo injection, we created and corrected chocolate missense mutations in the small GTPase RAB38; a regulator of intracellular vesicle trafficking and phenotypic model of Hermansky-Pudlak syndrome. Because ES cell cultures and targeting vectors are not required, this technology enables instant germline modifications, making heterozygous mutants available within 18 wk. The key features of direct mutagenesis by TALENs and oligodeoxynucleotides, minimal effort and high speed, catalyze the generation of future in vivo models for the study of human disease mechanisms and interventions. PMID:23426636

Functional and phenotypic characterization of CD8+CD28+ and CD28- T cells in atopic individuals sensitized to Dermatophagoides pteronyssinus.

PubMed

Lourenço, O; Fonseca, A M; Paiva, A; Arosa, F A; Taborda-Barata, L

2006-01-01

CD8+ T suppressor cells may play a role in immunoregulation. Recent studies have characterized this population by the lack of the CD28 molecule. These CD8+CD28 T cells differ phenotypically and functionally from CD8 + CD28 + T cells. Little is known about CD8 + CD28 cells in atopy. Our aim was to analyze the phenotype and functional properties of CD8 + CD28T cells in atopic and non-atopic individuals. Peripheral blood mononuclear cells (PBMC) were obtained after density gradient centrifugation. CD8 + CD28 and CD8 + CD28 + T cells were isolated using immunomagnetic beads. Relative percentages of these cells and expression of several phenotypic markers were analyzed by flow cytometry. Proliferation was assessed by thymidine incorporation in isolated populations and in co-cultures with PBMC using Dermatophagoides pteronyssinus as stimulus. Cytokine synthesis was evaluated in culture supernatants by cytometric bead array. The relative percentages of CD8+CD28 T cells and their phenotypic expression in atopic and non-atopic volunteers were not significantly different. However, CD8 + CD28 T cells showed greater proliferation than did CD8+CD28+ T cells when stimulated with D. pteronyssinus, although cytokine synthesis patterns were similar. CD8+CD28 co-cultures with PBMC showed greater proliferation than CD8+CD28+ T cell co-cultures, but cytokine synthesis patterns were not different. Our data confirm phenotypic and functional differences between CD28+ and CD28 T cells, irrespective of atopic status. Purified human CD8+CD28 T cells, freshly isolated from peripheral blood, do not have suppressor properties on allergen-specific proliferation or on cytokine synthesis in PBMC.

Effect of Apoptotic Cell Recognition on Macrophage Polarization and Mycobacterial Persistence

PubMed Central

de Oliveira Fulco, Tatiana; Andrade, Priscila Ribeiro; de Mattos Barbosa, Mayara Garcia; Pinto, Thiago Gomes Toledo; Ferreira, Paula Fernandez; Ferreira, Helen; da Costa Nery, José Augusto; Real, Suzana Côrte; Borges, Valéria Matos; Moraes, Milton Ozório; Sarno, Euzenir Nunes; Sampaio, Elizabeth Pereira

2014-01-01

Intracellular Mycobacterium leprae infection modifies host macrophage programming, creating a protective niche for bacterial survival. The milieu regulating cellular apoptosis in the tissue plays an important role in defining susceptible and/or resistant phenotypes. A higher density of apoptotic cells has been demonstrated in paucibacillary leprosy lesions than in multibacillary ones. However, the effect of apoptotic cell removal on M. leprae-stimulated cells has yet to be fully elucidated. In this study, we investigated whether apoptotic cell removal (efferocytosis) induces different phenotypes in proinflammatory (Mϕ1) and anti-inflammatory (Mϕ2) macrophages in the presence of M. leprae. We stimulated Mϕ1 and Mϕ2 cells with M. leprae in the presence or absence of apoptotic cells and subsequently evaluated the M. leprae uptake, cell phenotype, and cytokine pattern in the supernatants. In the presence of M. leprae and apoptotic cells, Mϕ1 macrophages changed their phenotype to resemble the Mϕ2 phenotype, displaying increased CD163 and SRA-I expression as well as higher phagocytic capacity. Efferocytosis increased M. leprae survival in Mϕ1 cells, accompanied by reduced interleukin-15 (IL-15) and IL-6 levels and increased transforming growth factor beta (TGF-β) and IL-10 secretion. Mϕ1 cells primed with M. leprae in the presence of apoptotic cells induced the secretion of Th2 cytokines IL-4 and IL-13 in autologous T cells compared with cultures stimulated with M. leprae or apoptotic cells alone. Efferocytosis did not alter the Mϕ2 cell phenotype or cytokine secretion profile, except for TGF-β. Based on these data, we suggest that, in paucibacillary leprosy patients, efferocytosis contributes to mycobacterial persistence by increasing the Mϕ2 population and sustaining the infection. PMID:25024361

Radiofrequency treatment alters cancer cell phenotype

NASA Astrophysics Data System (ADS)

Ware, Matthew J.; Tinger, Sophia; Colbert, Kevin L.; Corr, Stuart J.; Rees, Paul; Koshkina, Nadezhda; Curley, Steven; Summers, H. D.; Godin, Biana

2015-07-01

The importance of evaluating physical cues in cancer research is gradually being realized. Assessment of cancer cell physical appearance, or phenotype, may provide information on changes in cellular behavior, including migratory or communicative changes. These characteristics are intrinsically different between malignant and non-malignant cells and change in response to therapy or in the progression of the disease. Here, we report that pancreatic cancer cell phenotype was altered in response to a physical method for cancer therapy, a non-invasive radiofrequency (RF) treatment, which is currently being developed for human trials. We provide a battery of tests to explore these phenotype characteristics. Our data show that cell topography, morphology, motility, adhesion and division change as a result of the treatment. These may have consequences for tissue architecture, for diffusion of anti-cancer therapeutics and cancer cell susceptibility within the tumor. Clear phenotypical differences were observed between cancerous and normal cells in both their untreated states and in their response to RF therapy. We also report, for the first time, a transfer of microsized particles through tunneling nanotubes, which were produced by cancer cells in response to RF therapy. Additionally, we provide evidence that various sub-populations of cancer cells heterogeneously respond to RF treatment.

Neurological manifestations of oculodentodigital dysplasia: a Cx43 channelopathy of the central nervous system?

PubMed Central

De Bock, Marijke; Kerrebrouck, Marianne; Wang, Nan; Leybaert, Luc

2013-01-01

The coordination of tissue function is mediated by gap junctions (GJs) that enable direct cell–cell transfer of metabolic and electric signals. GJs are formed by connexins of which Cx43 is most widespread in the human body. In the brain, Cx43 GJs are mostly found in astroglia where they coordinate the propagation of Ca2+ waves, spatial K+ buffering, and distribution of glucose. Beyond its role in direct intercellular communication, Cx43 also forms unapposed, non-junctional hemichannels in the plasma membrane of glial cells. These allow the passage of several neuro- and gliotransmitters that may, combined with downstream paracrine signaling, complement direct GJ communication among glial cells and sustain glial-neuronal signaling. Mutations in the GJA1 gene encoding Cx43 have been identified in a rare, mostly autosomal dominant syndrome called oculodentodigital dysplasia (ODDD). ODDD patients display a pleiotropic phenotype reflected by eye, hand, teeth, and foot abnormalities, as well as craniofacial and bone malformations. Remarkably, neurological symptoms such as dysarthria, neurogenic bladder (manifested as urinary incontinence), spasticity or muscle weakness, ataxia, and epilepsy are other prominent features observed in ODDD patients. Over 10 mutations detected in patients diagnosed with neurological disorders are associated with altered functionality of Cx43 GJs/hemichannels, but the link between ODDD-related abnormal channel activities and neurologic phenotype is still elusive. Here, we present an overview on the nature of the mutants conveying structural and functional changes of Cx43 channels and discuss available evidence for aberrant Cx43 GJ and hemichannel function. In a final step, we examine the possibilities of how channel dysfunction may lead to some of the neurological manifestations of ODDD. PMID:24133447

Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes

PubMed Central

Baxter, Melissa; Withey, Sarah; Harrison, Sean; Segeritz, Charis-Patricia; Zhang, Fang; Atkinson-Dell, Rebecca; Rowe, Cliff; Gerrard, Dave T.; Sison-Young, Rowena; Jenkins, Roz; Henry, Joanne; Berry, Andrew A.; Mohamet, Lisa; Best, Marie; Fenwick, Stephen W.; Malik, Hassan; Kitteringham, Neil R.; Goldring, Chris E.; Piper Hanley, Karen; Vallier, Ludovic; Hanley, Neil A.

2015-01-01

Background & Aims Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells by the use of soluble factors, can model human liver function and toxicity. However, at present HLC maturity and whether any deficit represents a true fetal state or aberrant differentiation is unclear and compounded by comparison to potentially deteriorated adult hepatocytes. Therefore, we generated HLCs from multiple lineages, using two different protocols, for direct comparison with fresh fetal and adult hepatocytes. Methods Protocols were developed for robust differentiation. Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes. Results HLCs were comparable to those of other laboratories by multiple parameters. Transcriptional changes during differentiation mimicked human embryogenesis and showed more similarity to pericentral than periportal hepatocytes. Unbiased proteomics demonstrated greater proximity to liver than 30 other human organs or tissues. However, by comparison to fresh material, HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype. The expression of 81% phase 1 enzymes in HLCs was significantly upregulated and half were statistically not different from fetal hepatocytes. HLCs secreted albumin and metabolized testosterone (CYP3A) and dextrorphan (CYP2D6) like fetal hepatocytes. In seven bespoke tests, devised by principal components analysis to distinguish fetal from adult hepatocytes, HLCs from two different source laboratories consistently demonstrated fetal characteristics. Conclusions HLCs from different sources are broadly comparable with unbiased proteomic evidence for faithful differentiation down the liver lineage. This current phenotype mimics human fetal rather than adult hepatocytes. PMID:25457200

In vitro characterization of cancer cell morphology, chemokinesis, and matrix invasion using a novel microfabricated system

NASA Astrophysics Data System (ADS)

Blaha, Laura

A diagnosis of metastatic cancer reduces a patient's 5-year survival rate by nearly 80% compared to a primary tumor diagnosed at an early stage. While gene expression arrays have revealed unique gene signatures for metastatic cancer cells, we are lacking an understanding of the tangible physical changes that distinguish metastatic tumor cells from each other and from their related primary tumors. At the fundamental level, this translates into first characterizing the phenotype of metastatic cancer cells in vitro both in 2D - looking at morphology and migration - and in 3D - focusing on matrix invasion. While 2D in vitro studies have provided insight into the effects of specific environmental conditions on specific cancer cell lines, the unique details included in each experimental design make it challenging to compare cell phenotype across different in vitro platforms as well as between laboratories and disciplines that share the goal of understanding cancer. While 3D phenotype studies have employed more standardized and ubiquitous assays, most available tools lack the imaging capability and geometry to effectively characterize all factors driving 3D matrix invasion. In this work, we present protocols and platforms aimed at addressing the problems identified in the tools currently available for studying metastatic cancer in vitro. First, we present a 2D study of morphology and migration using widely accepted protocols. The study is applied to characterizing phenotypes of three breast cancer cell lines with different metastatic organ tropisms. The results show that general populations of cells from each of the 3 lines are unique in shape and motility despite being derived from the same tumor line and that the observed phenotype differences may be related to differences in focal adhesion assembly. More broadly, these studies suggest that standardizing phenotype studies using commonly available techniques may provide a platform by which to compare phenotypic studies across cancer cell types and between research groups to investigate tropism-specific cancer phenotypes. We conclude our investigation of phenotype with a study of 3D matrix invasion using a novel microfluidic platform. The results show that invasion of metastatic breast cancer cells into a 3D type I collagen gel is significantly enhanced in the presence of live endothelial cells. In applying the model to study cell-cell and cell-matrix interactions driving invasion, our platform revealed that, while the fibronectin-rich matrix deposited by endothelial cells was not sufficient to drive invasion alone, metastatic breast cancer cells were able to exploit a structural or secreted component of energetically inactivated endothelial cell to gain entry into the underlying matrix. These findings have important implications for designing drugs targeted at preventing cancer metastasis. The findings in this dissertation reveal significant phenotypic differences in metastatic breast cancer cells with different preferences in metastatic target organ. In addition, the microfluidic platform reveals novel cell-cell interactions driving a key step in the seeding and colonization of a metastatic tumor. Collectively, these results reveal important characteristics of metastatic cancer cells and their interactions with other cell types during metastasis. These studies also provide platforms on which to target or prevent malignant phenotypes and cellular interactions in the future.

Attrition of memory CD8 T cells during sepsis requires LFA-1.

PubMed

Serbanescu, Mara A; Ramonell, Kimberly M; Hadley, Annette; Margoles, Lindsay M; Mittal, Rohit; Lyons, John D; Liang, Zhe; Coopersmith, Craig M; Ford, Mandy L; McConnell, Kevin W

2016-11-01

CD8 T cell loss and dysfunction have been implicated in the increased susceptibility to opportunistic infections during the later immunosuppressive phase of sepsis, but CD8 T cell activation and attrition in early sepsis remain incompletely understood. With the use of a CLP model, we assessed CD8 T cell activation at 5 consecutive time points and found that activation after sepsis results in a distinct phenotype (CD69 + CD25 int CD62L HI ) independent of cognate antigen recognition and TCR engagement and likely through bystander-mediated cytokine effects. Additionally, we observed that sepsis concurrently results in the preferential depletion of a subset of memory-phenotype CD8 T cells that remain "unactivated" (i.e., fail to up-regulate activation markers) by apoptosis. Unactivated CD44 HI OT-I cells were spared from sepsis-induced attrition, as were memory-phenotype CD8 T cells of mice treated with anti-LFA-1 mAb, 1 h after CLP. Perhaps most importantly, we demonstrate that attrition of memory phenotype cells may have a pathologic significance, as elevated IL-6 levels were associated with decreased numbers of memory-phenotype CD8 T cells in septic mice, and preservation of this subset after administration of anti-LFA-1 mAb conferred improved survival at 7 d. Taken together, these data identify potentially modifiable responses of memory-phenotype CD8 T cells in early sepsis and may be particularly important in the application of immunomodulatory therapies in sepsis. © Society for Leukocyte Biology.

Molecular crosstalk between tumour and brain parenchyma instructs histopathological features in glioblastoma.

PubMed

Bougnaud, Sébastien; Golebiewska, Anna; Oudin, Anaïs; Keunen, Olivier; Harter, Patrick N; Mäder, Lisa; Azuaje, Francisco; Fritah, Sabrina; Stieber, Daniel; Kaoma, Tony; Vallar, Laurent; Brons, Nicolaas H C; Daubon, Thomas; Miletic, Hrvoje; Sundstrøm, Terje; Herold-Mende, Christel; Mittelbronn, Michel; Bjerkvig, Rolf; Niclou, Simone P

2016-05-31

The histopathological and molecular heterogeneity of glioblastomas represents a major obstacle for effective therapies. Glioblastomas do not develop autonomously, but evolve in a unique environment that adapts to the growing tumour mass and contributes to the malignancy of these neoplasms. Here, we show that patient-derived glioblastoma xenografts generated in the mouse brain from organotypic spheroids reproducibly give rise to three different histological phenotypes: (i) a highly invasive phenotype with an apparent normal brain vasculature, (ii) a highly angiogenic phenotype displaying microvascular proliferation and necrosis and (iii) an intermediate phenotype combining features of invasion and vessel abnormalities. These phenotypic differences were visible during early phases of tumour development suggesting an early instructive role of tumour cells on the brain parenchyma. Conversely, we found that tumour-instructed stromal cells differentially influenced tumour cell proliferation and migration in vitro, indicating a reciprocal crosstalk between neoplastic and non-neoplastic cells. We did not detect any transdifferentiation of tumour cells into endothelial cells. Cell type-specific transcriptomic analysis of tumour and endothelial cells revealed a strong phenotype-specific molecular conversion between the two cell types, suggesting co-evolution of tumour and endothelial cells. Integrative bioinformatic analysis confirmed the reciprocal crosstalk between tumour and microenvironment and suggested a key role for TGFβ1 and extracellular matrix proteins as major interaction modules that shape glioblastoma progression. These data provide novel insight into tumour-host interactions and identify novel stroma-specific targets that may play a role in combinatorial treatment strategies against glioblastoma.

Molecular crosstalk between tumour and brain parenchyma instructs histopathological features in glioblastoma

PubMed Central

Bougnaud, Sébastien; Golebiewska, Anna; Oudin, Anaïs; Keunen, Olivier; Harter, Patrick N.; Mäder, Lisa; Azuaje, Francisco; Fritah, Sabrina; Stieber, Daniel; Kaoma, Tony; Vallar, Laurent; Brons, Nicolaas H.C.; Daubon, Thomas; Miletic, Hrvoje; Sundstrøm, Terje; Herold-Mende, Christel; Mittelbronn, Michel; Bjerkvig, Rolf; Niclou, Simone P.

2016-01-01

The histopathological and molecular heterogeneity of glioblastomas represents a major obstacle for effective therapies. Glioblastomas do not develop autonomously, but evolve in a unique environment that adapts to the growing tumour mass and contributes to the malignancy of these neoplasms. Here, we show that patient-derived glioblastoma xenografts generated in the mouse brain from organotypic spheroids reproducibly give rise to three different histological phenotypes: (i) a highly invasive phenotype with an apparent normal brain vasculature, (ii) a highly angiogenic phenotype displaying microvascular proliferation and necrosis and (iii) an intermediate phenotype combining features of invasion and vessel abnormalities. These phenotypic differences were visible during early phases of tumour development suggesting an early instructive role of tumour cells on the brain parenchyma. Conversely, we found that tumour-instructed stromal cells differentially influenced tumour cell proliferation and migration in vitro, indicating a reciprocal crosstalk between neoplastic and non-neoplastic cells. We did not detect any transdifferentiation of tumour cells into endothelial cells. Cell type-specific transcriptomic analysis of tumour and endothelial cells revealed a strong phenotype-specific molecular conversion between the two cell types, suggesting co-evolution of tumour and endothelial cells. Integrative bioinformatic analysis confirmed the reciprocal crosstalk between tumour and microenvironment and suggested a key role for TGFβ1 and extracellular matrix proteins as major interaction modules that shape glioblastoma progression. These data provide novel insight into tumour-host interactions and identify novel stroma-specific targets that may play a role in combinatorial treatment strategies against glioblastoma. PMID:27049916

Attrition of memory CD8 T cells during sepsis requires LFA-1

PubMed Central

Serbanescu, Mara A.; Ramonell, Kimberly M.; Hadley, Annette; Margoles, Lindsay M.; Mittal, Rohit; Lyons, John D.; Liang, Zhe; Coopersmith, Craig M.; Ford, Mandy L.; McConnell, Kevin W.

2016-01-01

CD8 T cell loss and dysfunction have been implicated in the increased susceptibility to opportunistic infections during the later immunosuppressive phase of sepsis, but CD8 T cell activation and attrition in early sepsis remain incompletely understood. With the use of a CLP model, we assessed CD8 T cell activation at 5 consecutive time points and found that activation after sepsis results in a distinct phenotype (CD69+CD25intCD62LHI) independent of cognate antigen recognition and TCR engagement and likely through bystander-mediated cytokine effects. Additionally, we observed that sepsis concurrently results in the preferential depletion of a subset of memory-phenotype CD8 T cells that remain “unactivated” (i.e., fail to up-regulate activation markers) by apoptosis. Unactivated CD44HI OT-I cells were spared from sepsis-induced attrition, as were memory-phenotype CD8 T cells of mice treated with anti-LFA-1 mAb, 1 h after CLP. Perhaps most importantly, we demonstrate that attrition of memory phenotype cells may have a pathologic significance, as elevated IL-6 levels were associated with decreased numbers of memory-phenotype CD8 T cells in septic mice, and preservation of this subset after administration of anti-LFA-1 mAb conferred improved survival at 7 d. Taken together, these data identify potentially modifiable responses of memory-phenotype CD8 T cells in early sepsis and may be particularly important in the application of immunomodulatory therapies in sepsis. PMID:27286793

Design of experiments approach to engineer cell-secreted matrices for directing osteogenic differentiation.

PubMed

Decaris, Martin L; Leach, J Kent

2011-04-01

The presentation of extracellular matrix (ECM) proteins provides an opportunity to instruct the phenotype and behavior of responsive cells. Decellularized cell-secreted matrix coatings (DM) represent a biomimetic culture surface that retains the complexity of the natural ECM. Microenvironmental culture conditions alter the composition of these matrices and ultimately the ability of DMs to direct cell fate. We employed a design of experiments (DOE) multivariable analysis approach to determine the effects and interactions of four variables (culture duration, cell seeding density, oxygen tension, and media supplementation) on the capacity of DMs to direct the osteogenic differentiation of human mesenchymal stem cells (hMSCs). DOE analysis revealed that matrices created with extended culture duration, ascorbate-2-phosphate supplementation, and in ambient oxygen tension exhibited significant correlations with enhanced hMSC differentiation. We validated the DOE model results using DMs predicted to have superior (DM1) or lesser (DM2) osteogenic potential for naïve hMSCs. Compared to cells on DM2, hMSCs cultured on DM1 expressed 2-fold higher osterix levels and deposited 3-fold more calcium over 3 weeks. Cells on DM1 coatings also exhibited greater proliferation and viability compared to DM2-coated substrates. This study demonstrates that DOE-based analysis is a powerful tool for optimizing engineered systems by identifying significant variables that have the greatest contribution to the target output.

Toward understanding of the role of reversibility of phenotypic switching in the evolution of resistance to therapy

NASA Astrophysics Data System (ADS)

Horvath, D.; Brutovsky, B.

2018-06-01

Reversibility of state transitions is intensively studied topic in many scientific disciplines over many years. In cell biology, it plays an important role in epigenetic variation of phenotypes, known as phenotypic plasticity. More interestingly, the cell state reversibility is probably crucial in the adaptation of population phenotypic heterogeneity to environmental fluctuations by evolving bet-hedging strategy, which might confer to cancer cells resistance to therapy. In this article, we propose a formalization of the evolution of highly reversible states in the environments of periodic variability. Two interrelated models of heterogeneous cell populations are proposed and their behavior is studied. The first model captures selection dynamics of the cell clones for the respective levels of phenotypic reversibility. The second model focuses on the interplay between reversibility and drug resistance in the particular case of cancer. Overall, our results show that the threshold dependencies are emergent features of the investigated model with eventual therapeutic relevance. Presented examples demonstrate importance of taking into account cell to cell heterogeneity within a system of clones with different reversibility quantified by appropriately chosen genetic and epigenetic entropy measures.

Inflammatory cell phenotypes in AAAs: their role and potential as targets for therapy.

PubMed

Dale, Matthew A; Ruhlman, Melissa K; Baxter, B Timothy

2015-08-01

Abdominal aortic aneurysms (AAAs) are characterized by chronic inflammatory cell infiltration. AAA is typically an asymptomatic disease and caused ≈15 000 deaths annually in the United States. Previous studies have examined both human and murine aortic tissue for the presence of various inflammatory cell types. Studies show that in both human and experimental AAAs, prominent inflammatory cell infiltration, such as CD4(+) T cells and macrophages, occurs in the damaged aortic wall. These cells have the ability to undergo phenotypic modulation based on microenvironmental cues, potentially influencing disease progression. Proinflammatory CD4(+) T cells and classically activated macrophages dominate the landscape of aortic infiltrates. The skew to proinflammatory phenotypes alters disease progression and plays a role in causing chronic inflammation. The local cytokine production and presence of inflammatory mediators, such as extracellular matrix breakdown products, influence the uneven balance of the inflammatory infiltrate phenotypes. Understanding and developing new strategies that target the proinflammatory phenotype could provide useful therapeutic targets for a disease with no current pharmacological intervention. © 2015 American Heart Association, Inc.

Inflammatory cell phenotypes in AAAs; their role and potential as targets for therapy

PubMed Central

Dale, Matthew A; Ruhlman, Melissa K.; Baxter, B. Timothy

2015-01-01

Abdominal aortic aneurysms are characterized by chronic inflammatory cell infiltration. AAA is typically an asymptomatic disease and caused approximately 15,000 deaths annually in the U.S. Previous studies have examined both human and murine aortic tissue for the presence of various inflammatory cell types. Studies show that in both human and experimental AAAs, prominent inflammatory cell infiltration, such as CD4+ T cells and macrophages, occurs in the damaged aortic wall. These cells have the ability to undergo phenotypic modulation based on microenvironmental cues, potentially influencing disease progression. Pro-inflammatory CD4+ T cells and classically activated macrophages dominate the landscape of aortic infiltrates. The skew to pro-inflammatory phenotypes alters disease progression and plays a role in causing chronic inflammation. The local cytokine production and presence of inflammatory mediators, such as extracellular matrix breakdown products, influence the uneven balance of the inflammatory infiltrate phenotypes. Understanding and developing new strategies that target the pro-inflammatory phenotype could provide useful therapeutic targets for a disease with no current pharmacological intervention. PMID:26044582

Differences in adhesion of Candida albicans 3153A cells exhibiting switch phenotypes to buccal epithelium and stratum corneum.

PubMed

Vargas, K; Wertz, P W; Drake, D; Morrow, B; Soll, D R

1994-04-01

Cells of the laboratory strain 3153A of Candida albicans can be stimulated to undergo high-frequency phenotypic switching by a low dose of UV. We have compared the adhesive properties of cells exhibiting the basic original smooth (o-smooth) phenotype and three switch phenotypes (star, irregular wrinkle, and revertant smooth) to buccal epithelium and stratum corneum. The generalized hierarchy of adhesion is as follows: o-smooth > irregular wrinkle > revertant smooth > star. This is the inverse of the hierarchy of the proportions of elongate hyphae formed by these phenotypes in culture. These results suggest that the differences in adhesion between o-smooth and the three switch phenotypes of strain 3153A reflect, at least in part, the level of interference due to the formation of elongate hyphae, which tend to cause clumping in suspension. No major differences in the levels of adhesion of cells of the different phenotypes between buccal epithelium and stratum corneum were observed. Results which demonstrate that buccal epithelium induces germination (hypha formation) by conditioning the medium are also presented.

The ROCK isoforms differentially regulate the morphological characteristics of carcinoma cells.

PubMed

Jerrell, Rachel J; Leih, Mitchell J; Parekh, Aron

2017-06-26

Rho-associated kinase (ROCK) activity drives cell migration via actomyosin contractility. During invasion, individual cancer cells can transition between 2 modes of migration, mesenchymal and amoeboid. Changes in ROCK activity can cause a switch between these migration phenotypes which are defined by distinct morphologies. However, recent studies have shown that the ROCK isoforms are not functionally redundant as previously thought. Therefore, it is unclear whether the ROCK isoforms play different roles in regulating migration phenotypes. Here, we found that ROCK1 and ROCK2 differentially regulate carcinoma cell morphology resulting in intermediate phenotypes that share some mesenchymal and amoeboid characteristics. These findings suggest that the ROCK isoforms play unique roles in the phenotypic plasticity of mesenchymal carcinoma cells which may have therapeutic implications.

Functional Genomic Analysis of the let-7 Regulatory Network in Caenorhabditis elegans

PubMed Central

Zisoulis, Dimitrios G.; Lovci, Michael T.; Melnik-Martinez, Katya V.; Yeo, Gene W.; Pasquinelli, Amy E.

2013-01-01

The let-7 microRNA (miRNA) regulates cellular differentiation across many animal species. Loss of let-7 activity causes abnormal development in Caenorhabditis elegans and unchecked cellular proliferation in human cells, which contributes to tumorigenesis. These defects are due to improper expression of protein-coding genes normally under let-7 regulation. While some direct targets of let-7 have been identified, the genome-wide effect of let-7 insufficiency in a developing animal has not been fully investigated. Here we report the results of molecular and genetic assays aimed at determining the global network of genes regulated by let-7 in C. elegans. By screening for mis-regulated genes that also contribute to let-7 mutant phenotypes, we derived a list of physiologically relevant potential targets of let-7 regulation. Twenty new suppressors of the rupturing vulva or extra seam cell division phenotypes characteristic of let-7 mutants emerged. Three of these genes, opt-2, prmt-1, and T27D12.1, were found to associate with Argonaute in a let-7–dependent manner and are likely novel direct targets of this miRNA. Overall, a complex network of genes with various activities is subject to let-7 regulation to coordinate developmental timing across tissues during worm development. PMID:23516374

Stable knockdown of PASG enhances DNA demethylation but does not accelerate cellular senescence in TIG-7 human fibroblasts

PubMed Central

Suzuki, Toshikazu; Farrar, Jason E.; Yegnasubramanian, Srinivasan; Zahed, Muhammed; Suzuki, Nobuo; Arceci, Robert J.

2009-01-01

Demethylation of 5-methylcytosine in genomic DNA is believed to be one of the mechanisms underlying replicative life-span of mammalian cells. Both proliferation associated SNF2-like gene (PASG, also termed Lsh) and DNA methyltransferase 3B (Dnmt3b) knockout mice result in embryonic genomic hypomethylation and a replicative senescent phenotype. However, it is unclear whether gradual demethylation of DNA during somatic cell division is directly involved in senescence. In this study, we retrovirally transduced TIG-7 human fibroblasts with a shRNA against PASG and compared the rate of change in DNA methylation as well as the replicative life-span to control cells under low (3%) and ambient (20%) oxygen. Expression of PASG protein was decreased by approximately 80% compared to control cells following transduction of PASG shRNA gene. The rate of cell growth was the same in both control and PASG-suppressed cells. The rate of demethylation of DNA was significantly increased in PASG-suppressed cells as compared control cells. However, decreased PASG expression did not shorten the replicative life-span of TIG-7 cells. Culture under low oxygen extended the life-span of TIG-7 cells but did not alter the rate of DNA demethylation. While knockout of PASG during development results in genomic hypomethylation and premature senescence, our results show that while downregulation of PASG expression in a somatic cell also leads to DNA hypomethylation, there is no associated senescent phenotype. These results suggest differences in cellular consequences of hypomethylation mediated by PASG during development compared to that in somatic cells. PMID:18948754

Stable knockdown of PASG enhances DNA demethylation but does not accelerate cellular senescence in TIG-7 human fibroblasts.

PubMed

Suzuki, Toshikazu; Farrar, Jason E; Yegnasubramanian, Srinivasan; Zahed, Muhammed; Suzuki, Nobuo; Arceci, Robert J

2008-09-01

Demethylation of 5-methylcytosine in genomic DNA is believed to be one of the mechanisms underlying replicative life-span of mammalian cells. Both proliferation associated SNF2-like gene (PASG, also termed Lsh) and DNA methyltransferase 3B (Dnmt3b) knockout mice result in embryonic genomic hypomethylation and a replicative senescent phenotype. However, it is unclear whether gradual demethylation of DNA during somatic cell division is directly involved in senescence. In this study, we retrovirally transduced TIG-7 human fibroblasts with a shRNA against PASG and compared the rate of change in DNA methylation as well as the replicative life-span to control cells under low (3%) and ambient (20%) oxygen. Expression of PASG protein was decreased by approximately 80% compared to control cells following transduction of PASG shRNA gene. The rate of cell growth was the same in both control and PASG-suppressed cells. The rate of demethylation of DNA was significantly increased in PASG-suppressed cells as compared control cells. However, decreased PASG expression did not shorten the replicative life-span of TIG-7 cells. Culture under low oxygen extended the life-span of TIG-7 cells but did not alter the rate of DNA demethylation. While knockout of PASG during development results in genomic hypomethylation and premature senescence, our results show that while downregulation of PASG expression in a somatic cell also leads to DNA hypomethylation, there is no associated senescent phenotype. These results suggest differences in cellular consequences of hypomethylation mediated by PASG during development compared to that in somatic cells.

Network motifs that stabilize the hybrid epithelial/mesenchymal phenotype

NASA Astrophysics Data System (ADS)

Jolly, Mohit Kumar; Jia, Dongya; Tripathi, Satyendra; Hanash, Samir; Mani, Sendurai; Ben-Jacob, Eshel; Levine, Herbert

Epithelial to Mesenchymal Transition (EMT) and its reverse - MET - are hallmarks of cancer metastasis. While transitioning between E and M phenotypes, cells can also attain a hybrid epithelial/mesenchymal (E/M) phenotype that enables collective cell migration as a cluster of Circulating Tumor Cells (CTCs). These clusters can form 50-times more tumors than individually migrating CTCs, underlining their importance in metastasis. However, this hybrid E/M phenotype has been hypothesized to be only a transient one that is attained en route EMT. Here, via mathematically modeling, we identify certain `phenotypic stability factors' that couple with the core three-way decision-making circuit (miR-200/ZEB) and can maintain or stabilize the hybrid E/M phenotype. Further, we show experimentally that this phenotype can be maintained stably at a single-cell level, and knockdown of these factors impairs collective cell migration. We also show that these factors enable the association of hybrid E/M with high stemness or tumor-initiating potential. Finally, based on these factors, we deduce specific network motifs that can maintain the E/M phenotype. Our framework can be used to elucidate the effect of other players in regulating cellular plasticity during metastasis. This work was supported by NSF PHY-1427654 (Center for Theoretical Biological Physics) and the CPRIT Scholar in Cancer Research of the State of Texas at Rice University.

Plasticity of male germline stem cells and their applications in reproductive and regenerative medicine.

PubMed

Chen, Zheng; Li, Zheng; He, Zuping

2015-01-01

Spermatogonial stem cells (SSCs), also known as male germline stem cells, are a small subpopulation of type A spermatogonia with the potential of self-renewal to maintain stem cell pool and differentiation into spermatids in mammalian testis. SSCs are previously regarded as the unipotent stem cells since they can only give rise to sperm within the seminiferous tubules. However, this concept has recently been challenged because numerous studies have demonstrated that SSCs cultured with growth factors can acquire pluripotency to become embryonic stem-like cells. The in vivo and in vitro studies from peers and us have clearly revealed that SSCs can directly transdifferentiate into morphologic, phenotypic, and functional cells of other lineages. Direct conversion to the cells of other tissues has important significance for regenerative medicine. SSCs from azoospermia patients could be induced to differentiate into spermatids with fertilization and developmental potentials. As such, SSCs could have significant applications in both reproductive and regenerative medicine due to their unique and great potentials. In this review, we address the important plasticity of SSCs, with focuses on their self-renewal, differentiation, dedifferentiation, transdifferentiation, and translational medicine studies.

The Distinctive Sensitivity to Microgravity of Immune Cell Subpopulations

NASA Astrophysics Data System (ADS)

Chen, Hui; Luo, Haiying; Liu, Jing; Wang, Peng; Dong, Dandan; Shang, Peng; Zhao, Yong

2015-11-01

Immune dysfunction in astronauts is well documented after spaceflights. Microgravity is one of the key factors directly suppressing the function of immune system. However, it is unclear which subpopulations of immune cells including innate and adaptive immune cells are more sensitive to microgravity We herein investigated the direct effects of modeled microgravity (MMg) on different immune cells in vitro. Mouse splenocytes, thymocytes and bone marrow cells were exposed to MMg for 16 hrs. The survival and the phenotypes of different subsets of immune cells including CD4+T cells, CD8+T cells, CD4+Foxp3+ regulatory T cells (Treg), B cells, monocytes/macrophages, dendritic cells (DCs), natural killer cells (NK) were determined by flow cytometry. After splenocytes were cultured under MMg for 16h, the cell frequency and total numbers of monocytes, macrophages and CD4+Foxp3+T cells were significantly decreased more than 70 %. MMg significantly decreased the cell numbers of CD8+ T cells, B cells and neutrophils in splenocytes. The cell numbers of CD4+T cells and NK cells were unchanged significantly when splenocytes were cultured under MMg compared with controls. However, MMg significantly increased the ratio of mature neutrophils to immature neutrophils in bone marrow and the cell number of DCs in splenocytes. Based on the cell survival ability, monocytes, macrophages and CD4+Foxp3+Treg cells are most sensitive to microgravity; CD4+T cells and NK cells are resistant to microgravity; CD8+T cells and neutrophils are impacted by short term microgravity exposure. Microgravity promoted the maturation of neutrophils and development of DCs in vitro. The present studies offered new insights on the direct effects of MMg on the survival and homeostasis of immune cell subsets.

New approaches to the representation and analysis of phenotype knowledge in human diseases and their animal models.

PubMed

Schofield, Paul N; Sundberg, John P; Hoehndorf, Robert; Gkoutos, Georgios V

2011-09-01

The systematic investigation of the phenotypes associated with genotypes in model organisms holds the promise of revealing genotype-phenotype relations directly and without additional, intermediate inferences. Large-scale projects are now underway to catalog the complete phenome of a species, notably the mouse. With the increasing amount of phenotype information becoming available, a major challenge that biology faces today is the systematic analysis of this information and the translation of research results across species and into an improved understanding of human disease. The challenge is to integrate and combine phenotype descriptions within a species and to systematically relate them to phenotype descriptions in other species, in order to form a comprehensive understanding of the relations between those phenotypes and the genotypes involved in human disease. We distinguish between two major approaches for comparative phenotype analyses: the first relies on evolutionary relations to bridge the species gap, while the other approach compares phenotypes directly. In particular, the direct comparison of phenotypes relies heavily on the quality and coherence of phenotype and disease databases. We discuss major achievements and future challenges for these databases in light of their potential to contribute to the understanding of the molecular mechanisms underlying human disease. In particular, we discuss how the use of ontologies and automated reasoning can significantly contribute to the analysis of phenotypes and demonstrate their potential for enabling translational research.

The phenotypic equilibrium of cancer cells: From average-level stability to path-wise convergence.

PubMed

Niu, Yuanling; Wang, Yue; Zhou, Da

2015-12-07

The phenotypic equilibrium, i.e. heterogeneous population of cancer cells tending to a fixed equilibrium of phenotypic proportions, has received much attention in cancer biology very recently. In the previous literature, some theoretical models were used to predict the experimental phenomena of the phenotypic equilibrium, which were often explained by different concepts of stabilities of the models. Here we present a stochastic multi-phenotype branching model by integrating conventional cellular hierarchy with phenotypic plasticity mechanisms of cancer cells. Based on our model, it is shown that: (i) our model can serve as a framework to unify the previous models for the phenotypic equilibrium, and then harmonizes the different kinds of average-level stabilities proposed in these models; and (ii) path-wise convergence of our model provides a deeper understanding to the phenotypic equilibrium from stochastic point of view. That is, the emergence of the phenotypic equilibrium is rooted in the stochastic nature of (almost) every sample path, the average-level stability just follows from it by averaging stochastic samples. Copyright © 2015 Elsevier Ltd. All rights reserved.

Introduction to cell–hydrogel mechanosensing

PubMed Central

Ahearne, Mark

2014-01-01

The development of hydrogel-based biomaterials represents a promising approach to generating new strategies for tissue engineering and regenerative medicine. In order to develop more sophisticated cell-seeded hydrogel constructs, it is important to understand how cells mechanically interact with hydrogels. In this paper, we review the mechanisms by which cells remodel hydrogels, the influence that the hydrogel mechanical and structural properties have on cell behaviour and the role of mechanical stimulation in cell-seeded hydrogels. Cell-mediated remodelling of hydrogels is directed by several cellular processes, including adhesion, migration, contraction, degradation and extracellular matrix deposition. Variations in hydrogel stiffness, density, composition, orientation and viscoelastic characteristics all affect cell activity and phenotype. The application of mechanical force on cells encapsulated in hydrogels can also instigate changes in cell behaviour. By improving our understanding of cell–material mechano-interactions in hydrogels, this should enable a new generation of regenerative medical therapies to be developed. PMID:24748951

A central role for vesicle trafficking in epithelial neoplasia: Intracellular highways to carcinogenesis

PubMed Central

Goldenring, James R.

2014-01-01

Epithelial cell carcinogenesis involves the loss of polarity, alteration of polarized protein presentation, dynamic cell morphology changes, increased proliferation and increased cell motility and invasion. Elements of membrane vesicle trafficking underlie all of these processes. Specific membrane trafficking regulators, including Rab small GTPases, through the coordinated dynamics of intracellular trafficking along cytoskeletal pathways, determine cell surface presentation of proteins and overall function of both differentiated and neoplastic cells. While mutations in vesicle trafficking proteins may not be direct drivers of transformation, elements of the machinery of vesicle movement play critical roles in the phenotypes of neoplastic cells. Therefore, the regulators of membrane vesicle trafficking decisions are critical mediators of the full spectrum of cell physiologies driving cancer cell biology, including initial loss of polarity, invasion and metastasis. Targeting of these fundamental intracellular processes may provide important points for manipulation of cancer cell behaviour. PMID:24108097

Direct transdifferentiation of spermatogonial stem cells to morphological, phenotypic and functional hepatocyte-like cells via the ERK1/2 and Smad2/3 signaling pathways and the inactivation of cyclin A, cyclin B and cyclin E

PubMed Central

2013-01-01

Background Severe shortage of liver donors and hepatocytes highlights urgent requirement of extra-liver and stem cell source of hepatocytes for treating liver-related diseases. Here we hypothesized that spermatogonial stem cells (SSCs) can directly transdifferentiate to hepatic stem-like cells capable of differentiating into mature hepatocyte-like cells in vitro without an intervening pluripotent state. Results SSCs first changed into hepatic stem-like cells since they resembled hepatic oval cells in morphology and expressed Ck8, Ck18, Ck7, Ck19, OV6, and albumin. Importantly, they co-expressed CK8 and CK19 but not ES cell markers. Hepatic stem-like cells derived from SSCs could differentiate into small hepatocytes based upon their morphological features and expression of numerous hepatic cell markers but lacking of bile epithelial cell hallmarks. Small hepatocytes were further coaxed to differentiate into mature hepatocyte-like cells, as identified by their morphological traits and strong expression of Ck8, Ck18, Cyp7a1, Hnf3b, Alb, Tat, Ttr, albumin, and CYP1A2 but not Ck7 or CK19. Notably, these differentiated cells acquired functional attributes of hepatocyte-like cells because they secreted albumin, synthesized urea, and uptake and released indocyanine green. Moreover, phosphorylation of ERK1/2 and Smad2/3 rather than Akt was activated in hepatic stem cells and mature hepatocytes. Additionally, cyclin A, cyclin B and cyclin E transcripts and proteins but not cyclin D1 or CDK1 and CDK2 transcripts or proteins were reduced in mature hepatocyte-like cells or hepatic stem-like cells derived from SSCs compared to SSCs. Conclusions SSCs can transdifferentiate to hepatic stem-like cells capable of differentiating into cells with morphological, phenotypic and functional characteristics of mature hepatocytes via the activation of ERK1/2 and Smad2/3 signaling pathways and the inactivation of cyclin A, cyclin B and cyclin E. This study thus provides an invaluable source of mature hepatocytes for treating liver-related diseases and drug toxicity screening and offers novel insights into mechanisms of liver development and cell reprogramming. PMID:24047406

RNAi-dependent and -independent antiviral phenotypes of chromosomally integrated shRNA clones: role of VASP in respiratory syncytial virus growth.

PubMed

Musiyenko, Alla; Bitko, Vira; Barik, Sailen

2007-07-01

Stable RNA interference (RNAi) is commonly achieved by recombinant expression of short hairpin RNA (shRNA). To generate virus-resistant cell lines, we cloned a shRNA cassette against the phosphoprotein gene of respiratory syncytial virus (RSV) into a polIII-driven plasmid vector. Analysis of individual stable transfectants showed a spectrum of RSV resistance correlating with the levels of shRNA expressed from different chromosomal locations. Interestingly, resistance in a minority of clones was due to mono-allelic disruption of the cellular gene for vasodilator-stimulated phosphoprotein (VASP). Thus, pure clones of chromosomally integrated DNA-directed RNAi can exhibit gene disruption phenotypes resembling but unrelated to RNAi.

GFP Labeling and Hepatic Differentiation Potential of Human Placenta-Derived Mesenchymal Stem Cells.

PubMed

Yu, Jiong; Su, Xiaoru; Zhu, Chengxing; Pan, Qiaoling; Yang, Jinfeng; Ma, Jing; Shen, Leyao; Cao, Hongcui; Li, Lanjuan

2015-01-01

Stem cell-based therapy in liver diseases has received increasing interest over the past decade, but direct evidence of the homing and implantation of transplanted cells is conflicting. Reliable labeling and tracking techniques are essential but lacking. The purpose of this study was to establish human placenta-derived mesenchymal stem cells (hPMSCs) expressing green fluorescent protein (GFP) and to assay their hepatic functional differentiation in vitro. The GFP gene was transduced into hPMSCs using a lentivirus to establish GFP(+) hPMSCs. GFP(+) hPMSCs were analyzed for their phenotypic profile, viability and adipogenic, osteogenic and hepatic differentiation. The derived GFP(+) hepatocyte-like cells were evaluated for their metabolic, synthetic and secretory functions, respectively. GFP(+) hPMSCs expressed high levels of HLA I, CD13, CD105, CD73, CD90, CD44 and CD29, but were negative for HLA II, CD45, CD31, CD34, CD133, CD271 and CD79. They possessed adipogenic, osteogenic and hepatic differentiation potential. Hepatocyte-like cells derived from GFP(+) hPMSCs showed typical hepatic phenotypes. GFP gene transduction has no adverse influences on the cellular or biochemical properties of hPMSCs or markers. GFP gene transduction using lentiviral vectors is a reliable labeling and tracking method. GFP(+) hPMSCs can therefore serve as a tool to investigate the mechanisms of MSC-based therapy, including hepatic disease therapy. © 2015 S. Karger AG, Basel.

Integrated Transcriptomic and Epigenomic Analysis of Primary Human Lung Epithelial Cell Differentiation

PubMed Central

Marconett, Crystal N.; Zhou, Beiyun; Rieger, Megan E.; Selamat, Suhaida A.; Dubourd, Mickael; Fang, Xiaohui; Lynch, Sean K.; Stueve, Theresa Ryan; Siegmund, Kimberly D.; Berman, Benjamin P.

2013-01-01

Elucidation of the epigenetic basis for cell-type specific gene regulation is key to gaining a full understanding of how the distinct phenotypes of differentiated cells are achieved and maintained. Here we examined how epigenetic changes are integrated with transcriptional activation to determine cell phenotype during differentiation. We performed epigenomic profiling in conjunction with transcriptomic profiling using in vitro differentiation of human primary alveolar epithelial cells (AEC). This model recapitulates an in vivo process in which AEC transition from one differentiated cell type to another during regeneration following lung injury. Interrogation of histone marks over time revealed enrichment of specific transcription factor binding motifs within regions of changing chromatin structure. Cross-referencing of these motifs with pathways showing transcriptional changes revealed known regulatory pathways of distal alveolar differentiation, such as the WNT and transforming growth factor beta (TGFB) pathways, and putative novel regulators of adult AEC differentiation including hepatocyte nuclear factor 4 alpha (HNF4A), and the retinoid X receptor (RXR) signaling pathways. Inhibition of the RXR pathway confirmed its functional relevance for alveolar differentiation. Our incorporation of epigenetic data allowed specific identification of transcription factors that are potential direct upstream regulators of the differentiation process, demonstrating the power of this approach. Integration of epigenomic data with transcriptomic profiling has broad application for the identification of regulatory pathways in other models of differentiation. PMID:23818859

Generation of a Gaussia luciferase-expressing endotheliotropic cytomegalovirus for screening approaches and mutant analyses.

PubMed

Falk, Jessica J; Laib Sampaio, Kerstin; Stegmann, Cora; Lieber, Diana; Kropff, Barbara; Mach, Michael; Sinzger, Christian

2016-09-01

For many questions in human cytomegalovirus (HCMV) research, assays are desired that allow robust and fast quantification of infection efficiencies under high-throughput conditions. The secreted Gaussia luciferase has been demonstrated as a suitable reporter in the context of a fibroblast-adapted HCMV strain, which however is greatly restricted in the number of cell types to which it can be applied. We inserted the Gaussia luciferase expression cassette into the BAC-cloned virus strain TB40-BAC4, which displays the natural broad cell tropism of HCMV and hence allows application to screening approaches in a variety of cell types including fibroblasts, epithelial, and endothelial cells. Here, we applied the reporter virus TB40-BAC4-IE-GLuc to identify mouse hybridoma clones that preferentially neutralize infection of endothelial cells. In addition, as the Gaussia luciferase is secreted into culture supernatants from infected cells it allows kinetic analyses in living cultures. This can speed up and facilitate phenotypic characterization of BAC-cloned mutants. For example, we analyzed a UL74 stop-mutant of TB40-BAC4-IE-GLuc immediately after reconstitution in transfected cultures and found the increase of luciferase delayed and reduced as compared to wild type. Phenotypic monitoring directly in transfected cultures can minimize the risk of compensating mutations that might occur with extended passaging. Copyright © 2016 Elsevier B.V. All rights reserved.

Familial urothelial cell carcinoma of the bladder with autosomal dominant inheritance and late onset phenotype.

PubMed

Brown, Robin; Donnelly, Deirdre E; Allen, Derek; Loughrey, Maurice B; Morrison, Patrick J

2014-01-01

Familial Urothelial cell bladder cancer is rare. We report two families with urothelial cell carcinoma (UCC) of bladder with family history in other relatives, displaying probable autosomal dominant inheritance and a late onset pure UCC phenotype, and document the phenotype in each family. Descriptive familial study on two pedigrees over three generations. Two families with UCC bladder were identified, and the phenotype documented, each family having three cases of late onset UCC. Some cases of UCC are hereditary and may display autosomal dominant inheritance with late onset of the cancer. Clinicians should be aware of the existence of a familial late onset UCC phenotype when managing cases of UCC.

The highly conserved MraZ protein is a transcriptional regulator in Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eraso, Jesus M.; Markillie, Lye Meng; Mitchell, Hugh D.

2014-05-05

The mraZ and mraW genes are highly conserved in bacteria, both in sequence and location at the head of the division and cell wall (dcw) gene cluster. Although MraZ has structural similarity to the AbrB transition state regulator and the MazE antitoxin, and MraW is known to methylate ribosomal RNA, mraZ and mraW null mutants have no detectable growth phenotype in any species tested to date, hampering progress in understanding their physiological role. Here we show that overproduction of Escherichia coli MraZ perturbs cell division and the cell envelope, is more lethal at high levels or in minimal growth medium,more » and that MraW antagonizes these effects. MraZGFP localizes to the nucleoid, suggesting that it binds DNA. Indeed, purified MraZ directly binds a region upstream from its own promoter containing three direct repeats to regulate its own expression and that of downstream cell division and cell wall genes. MraZ-LacZ fusions are repressed by excess MraZ but not when DNA binding by MraZ is inhibited. RNAseq analysis indicates that MraZ is a global transcriptional regulator with numerous targets in addition to dcw genes. One of these targets, mioC, is directly bound by MraZ in a region with three direct repeats.« less

Caspase-3 Is Transiently Activated without Cell Death during Early Antigen Driven Expansion of CD8+ T Cells In Vivo

PubMed Central

McComb, Scott; Mulligan, Rebecca; Sad, Subash

2010-01-01

Background CD8+ T cell responses develop rapidly during infection and are swiftly reduced during contraction, wherein >90% of primed CD8+ T cells are eliminated. The role of apoptotic mechanisms in controlling this rapid proliferation and contraction of CD8+ T cells remains unclear. Surprisingly, evidence has shown non-apoptotic activation of caspase-3 to occur during in vitro T-cell proliferation, but the relevance of these mechanisms to in vivo CD8+ T cell responses has yet to be examined. Methods and Findings We have evaluated the activity of caspase-3, a key downstream inducer of apoptosis, throughout the entirety of a CD8+ T cell response. We utilized two infection models that differ in the intensity, onset and duration of antigen-presentation and inflammation. Expression of cleaved caspase-3 in antigen specific CD8+ T cells was coupled to the timing and strength of antigen presentation in lymphoid organs. We also observed coordinated activation of additional canonical apoptotic markers, including phosphatidylserine exposure. Limiting dilution analysis directly showed that in the presence of IL7, very little cell death occurred in both caspase-3hi and caspase-3low CD8+ T cells. The expression of active caspase-3 peaked before effector phenotype (CD62Llow) CD8+ T cells emerged, and was undetectable in effector-phenotype cells. In addition, OVA-specific CD8+ cells remained active caspase-3low throughout the contraction phase. Conclusions Our results specifically implicate antigen and not inflammation in driving activation of apoptotic mechanisms without cell death in proliferating CD8+ T cells. Furthermore, the contraction of CD8+ T cell response following expansion is likely not mediated by the key downstream apoptosis inducer, caspase-3. PMID:21203525

Recent studies on the developing human hepatocellular carcinoma.

PubMed

Gerber, M A

1986-01-01

From our knowledge of characteristic phenotypic changes of the preneoplastic lesions during the stepwise evolution of hepatocellular carcinoma (HCC) in experimental models, we are now beginning to define the structural, histochemical, biochemical, antigenic and molecular properties of early HCC and of the putative preneoplastic changes in human liver. Histological, ultrastructural, morphometric and immunohistochemical studies suggest that adenomatous nodules of regenerating and hyperplastic hepatocytes are more likely to represent direct precursors of HCC than dysplastic hepatocytes. Histochemical and immunomorphological investigations show appreciable functional and phenotypic heterogeneity of human HCC as previously recognized in experimental hepatocarcinogenesis. Studies of altered expression of oncogenes in the regenerating liver and HCC are beginning to define the molecular mechanisms in cell growth and malignant transformation. Although integration of Hepadna viral DNA sequences frequently occurs during persistent infection in man and animals, the exact mechanism of viral oncogenesis remains to be elucidated. It is likely that the development of monoclonal antibodies to surface antigens on transformed hepatocytes will be useful for exploring lineage relationships between the cell populations involved in hepatocarcinogenesis.

Secondary ion mass spectrometry and Raman spectroscopy for tissue engineering applications

PubMed Central

Ilin, Yelena; Kraft, Mary L.

2014-01-01

Identifying the matrix properties that permit directing stem cell fate is critical for expanding desired cell lineages ex vivo for disease treatment. Such efforts require knowledge of matrix surface chemistry and the cell responses they elicit. Recent progress in analyzing biomaterial composition and identifying cell phenotype with two label-free chemical imaging techniques, TOF-SIMS and Raman spectroscopy are presented. TOF-SIMS is becoming indispensable for the surface characterization of biomaterial scaffolds. Developments in TOF-SIMS data analysis enable correlating surface chemistry with biological response. Advances in the interpretation of Raman spectra permit identifying the fate decisions of individual, living cells with location specificity. Here we highlight this progress and discuss further improvements that would facilitate efforts to develop artificial scaffolds for tissue regeneration. PMID:25462628

Endothelin and hepatic wound healing

PubMed Central

Khimji, Al-karim; Rockey, Don C.

2014-01-01

Liver wound healing is a coordinated response to injury caused by infections (hepatitis) or toxins (alcohol) or other processes where activation of hepatic stellate cells are a central component. During stellate cell activation, a major phenotypic transformation occurs which leads to increased production of increased extracellular matrix proteins and smooth muscle α-actin the results is organ dysfunction due to gross architectural disruption and impaired blood flow. Endothelin-1 (ET-1) is produced in increased amounts and the cellular source of ET-1 shifts from endothelial cells to stellate cells during liver injury thus setting a feedback loop which accentuates further activation, stellate cell proliferation, and production of extracellular matrix proteins. Therapy directed at intervening the ET-1 signaling pathway has significant therapeutic potential in patients with liver disease. PMID:21421048

Tumor evolution in space: the effects of competition colonization tradeoffs on tumor invasion dynamics.

PubMed

Orlando, Paul A; Gatenby, Robert A; Brown, Joel S

2013-01-01

We apply competition colonization tradeoff models to tumor growth and invasion dynamics to explore the hypothesis that varying selection forces will result in predictable phenotypic differences in cells at the tumor invasive front compared to those in the core. Spatially, ecologically, and evolutionarily explicit partial differential equation models of tumor growth confirm that spatial invasion produces selection pressure for motile phenotypes. The effects of the invasive phenotype on normal adjacent tissue determine the patterns of growth and phenotype distribution. If tumor cells do not destroy their environment, colonizer and competitive phenotypes coexist with the former localized at the invasion front and the latter, to the tumor interior. If tumors cells do destroy their environment, then cell motility is strongly selected resulting in accelerated invasion speed with time. Our results suggest that the widely observed genetic heterogeneity within cancers may not be the stochastic effect of random mutations. Rather, it may be the consequence of predictable variations in environmental selection forces and corresponding phenotypic adaptations.

Tumor Evolution in Space: The Effects of Competition Colonization Tradeoffs on Tumor Invasion Dynamics

PubMed Central

Orlando, Paul A.; Gatenby, Robert A.; Brown, Joel S.

2013-01-01

We apply competition colonization tradeoff models to tumor growth and invasion dynamics to explore the hypothesis that varying selection forces will result in predictable phenotypic differences in cells at the tumor invasive front compared to those in the core. Spatially, ecologically, and evolutionarily explicit partial differential equation models of tumor growth confirm that spatial invasion produces selection pressure for motile phenotypes. The effects of the invasive phenotype on normal adjacent tissue determine the patterns of growth and phenotype distribution. If tumor cells do not destroy their environment, colonizer and competitive phenotypes coexist with the former localized at the invasion front and the latter, to the tumor interior. If tumors cells do destroy their environment, then cell motility is strongly selected resulting in accelerated invasion speed with time. Our results suggest that the widely observed genetic heterogeneity within cancers may not be the stochastic effect of random mutations. Rather, it may be the consequence of predictable variations in environmental selection forces and corresponding phenotypic adaptations. PMID:23508890

Breast fibroblasts in both cancer and normal tissues induce phenotypic transformation of breast cancer stem cells: a preliminary study

PubMed Central

Xi, Chunfang; Liu, Mingwei; Sun, Haichen; Liu, Shuang; Song, Lei

2018-01-01

Background Breast cancer stem cells (BCSCs) are associated with the invasion of breast cancer. In recent years, studies have demonstrated different phenotypes among BCSCs. Furthermore, BCSCs of diverse phenotypes are present at different tumour sites and different histological stages. Fibroblasts are involved in the phenotypic transformation of BCSCs. Cancer-associated fibroblasts (CAFs) participate in the induction of epithelial–mesenchymal transition, thereby promoting the acquisition of stem cell characteristics, but little is known about the role of normal fibroblasts (NFs) in the phenotypic transformation of BCSCs or about the effect of CAFs and NFs on BCSC phenotypes. Methods A total of six pairs of primary CAFs and NFs were isolated from surgical samples of breast cancer patients and subjected to morphological, immunohistochemical, cell invasion and proteomics analyses. After establishing a cell culture system with conditioned medium from CAFs and NFs, we used the mammosphere formation assay to explore the effect of CAFs and NFs on the self-renewal ability of BCSCs. The effect of CAFs and NFs on the phenotypic differentiation of BCSCs was further analysed by flow cytometry and immunofluorescence. Results The isolated CAFs and NFs did not show significant differences in cell morphology or alpha-smooth muscle actin (α-SMA) expression, but cell invasion and proteomics analyses demonstrated heterogeneity among these fibroblasts. Both CAFs and NFs could promote the generation of BCSCs, but CAFs displayed a greater ability than NFs in promoting mammosphere formation. Conditioned medium from CAFs increased the proportion of aldehyde dehydrogenase-1 positive (ALDH1+) BCSCs, but conditioned medium from NFs was more likely to promote the generation of CD44+CD24− BCSCs from MCF-7 cells. Discussion This study validated the heterogeneity among CAFs and NFs and expanded on the conclusion that fibroblasts promote the generation of cancer stem cells. Our results particularly emphasized the effect of NFs on the phenotypic transformation of BCSCs. In addition, this study further highlighted the roles of CAFs and NFs in the induction of different phenotypes in BCSCs. PMID:29780673

Breast fibroblasts in both cancer and normal tissues induce phenotypic transformation of breast cancer stem cells: a preliminary study.

PubMed

Wang, Bixiao; Xi, Chunfang; Liu, Mingwei; Sun, Haichen; Liu, Shuang; Song, Lei; Kang, Hua

2018-01-01

Breast cancer stem cells (BCSCs) are associated with the invasion of breast cancer. In recent years, studies have demonstrated different phenotypes among BCSCs. Furthermore, BCSCs of diverse phenotypes are present at different tumour sites and different histological stages. Fibroblasts are involved in the phenotypic transformation of BCSCs. Cancer-associated fibroblasts (CAFs) participate in the induction of epithelial-mesenchymal transition, thereby promoting the acquisition of stem cell characteristics, but little is known about the role of normal fibroblasts (NFs) in the phenotypic transformation of BCSCs or about the effect of CAFs and NFs on BCSC phenotypes. A total of six pairs of primary CAFs and NFs were isolated from surgical samples of breast cancer patients and subjected to morphological, immunohistochemical, cell invasion and proteomics analyses. After establishing a cell culture system with conditioned medium from CAFs and NFs, we used the mammosphere formation assay to explore the effect of CAFs and NFs on the self-renewal ability of BCSCs. The effect of CAFs and NFs on the phenotypic differentiation of BCSCs was further analysed by flow cytometry and immunofluorescence. The isolated CAFs and NFs did not show significant differences in cell morphology or alpha-smooth muscle actin (α-SMA) expression, but cell invasion and proteomics analyses demonstrated heterogeneity among these fibroblasts. Both CAFs and NFs could promote the generation of BCSCs, but CAFs displayed a greater ability than NFs in promoting mammosphere formation. Conditioned medium from CAFs increased the proportion of aldehyde dehydrogenase-1 positive (ALDH1 + ) BCSCs, but conditioned medium from NFs was more likely to promote the generation of CD44 + CD24 - BCSCs from MCF-7 cells. This study validated the heterogeneity among CAFs and NFs and expanded on the conclusion that fibroblasts promote the generation of cancer stem cells. Our results particularly emphasized the effect of NFs on the phenotypic transformation of BCSCs. In addition, this study further highlighted the roles of CAFs and NFs in the induction of different phenotypes in BCSCs.

Detection of characteristic sub pathway network for angiogenesis based on the comprehensive pathway network.

PubMed

Huang, Yezhou; Li, Shao

2010-01-18

Pathways in biological system often cooperate with each other to function. Changes of interactions among pathways tightly associate with alterations in the properties and functions of the cell and hence alterations in the phenotype. So, the pathway interactions and especially their changes over time corresponding to specific phenotype are critical to understanding cell functions and phenotypic plasticity. With prior-defined pathways and incorporated protein-protein interaction (PPI) data, we counted PPIs between corresponding gene sets of each pair of distinct pathways to construct a comprehensive pathway network. Then we proposed a novel concept, characteristic sub pathway network (CSPN), to realize the phenotype-specific pathway interactions. By adding gene expression data regarding a given phenotype, angiogenesis, active PPIs corresponding to stimulation of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) on human umbilical vein endothelial cells (HUVECs) respectively were derived. Two kinds of CSPN, namely the static or the dynamic CSPN, were detected by counting active PPIs. A comprehensive pathway network containing 37 signalling pathways as nodes and 263 pathway interactions were obtained. Two phenotype-specific CSPNs for angiogenesis, corresponding to stimulation of IL-1 and TNF-alpha on HUVEC respectively, were addressed. From phenotype-specific CSPNs, a static CSPN involving interactions among B cell receptor, T cell receptor, Toll-like receptor, MAPK, VEGF, and ErbB signalling pathways, and a dynamic CSPN involving interactions among TGF-beta, Wnt, p53 signalling pathways and cell cycle pathway, were detected for angiogenesis on HUVEC after stimulation of IL-1 and TNF-alpha respectively. We inferred that, in certain case, the static CSPN maintains related basic functions of the cells, whereas the dynamic CSPN manifests the cells' plastic responses to stimulus and therefore reflects the cells' phenotypic plasticity. The comprehensive pathway network helps us realize the cooperative behaviours among pathways. Moreover, two kinds of potential CSPNs found in this work, the static CSPN and the dynamic CSPN, are helpful to deeply understand the specific function of HUVEC and its phenotypic plasticity in regard to angiogenesis.

Detection of characteristic sub pathway network for angiogenesis based on the comprehensive pathway network

PubMed Central

2010-01-01

Background Pathways in biological system often cooperate with each other to function. Changes of interactions among pathways tightly associate with alterations in the properties and functions of the cell and hence alterations in the phenotype. So, the pathway interactions and especially their changes over time corresponding to specific phenotype are critical to understanding cell functions and phenotypic plasticity. Methods With prior-defined pathways and incorporated protein-protein interaction (PPI) data, we counted PPIs between corresponding gene sets of each pair of distinct pathways to construct a comprehensive pathway network. Then we proposed a novel concept, characteristic sub pathway network (CSPN), to realize the phenotype-specific pathway interactions. By adding gene expression data regarding a given phenotype, angiogenesis, active PPIs corresponding to stimulation of interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α) on human umbilical vein endothelial cells (HUVECs) respectively were derived. Two kinds of CSPN, namely the static or the dynamic CSPN, were detected by counting active PPIs. Results A comprehensive pathway network containing 37 signalling pathways as nodes and 263 pathway interactions were obtained. Two phenotype-specific CSPNs for angiogenesis, corresponding to stimulation of IL-1 and TNF-α on HUVEC respectively, were addressed. From phenotype-specific CSPNs, a static CSPN involving interactions among B cell receptor, T cell receptor, Toll-like receptor, MAPK, VEGF, and ErbB signalling pathways, and a dynamic CSPN involving interactions among TGF-β, Wnt, p53 signalling pathways and cell cycle pathway, were detected for angiogenesis on HUVEC after stimulation of IL-1 and TNF-α respectively. We inferred that, in certain case, the static CSPN maintains related basic functions of the cells, whereas the dynamic CSPN manifests the cells' plastic responses to stimulus and therefore reflects the cells' phenotypic plasticity. Conclusion The comprehensive pathway network helps us realize the cooperative behaviours among pathways. Moreover, two kinds of potential CSPNs found in this work, the static CSPN and the dynamic CSPN, are helpful to deeply understand the specific function of HUVEC and its phenotypic plasticity in regard to angiogenesis. PMID:20122205

A Dual-Mode Bioreactor System for Tissue Engineered Vascular Models.

PubMed

Bono, N; Meghezi, S; Soncini, M; Piola, M; Mantovani, D; Fiore, Gianfranco Beniamino

2017-06-01

In the past decades, vascular tissue engineering has made great strides towards bringing engineered vascular tissues to the clinics and, in parallel, obtaining in-lab tools for basic research. Herein, we propose the design of a novel dual-mode bioreactor, useful for the fabrication (construct mode) and in vitro stimulation (culture mode) of collagen-based tubular constructs. Collagen-based gels laden with smooth muscle cells (SMCs) were molded directly within the bioreactor culture chamber. Based on a systematic characterization of the bioreactor culture mode, constructs were subjected to 10% cyclic strain at 0.5 Hz for 5 days. The effects of cyclic stimulation on matrix re-arrangement and biomechanical/viscoelastic properties were examined and compared vs. statically cultured constructs. A thorough comparison of cell response in terms of cell localization and expression of contractile phenotypic markers was carried out as well. We found that cyclic stimulation promoted cell-driven collagen matrix bi-axial compaction, enhancing the mechanical strength of strained samples with respect to static controls. Moreover, cyclic strain positively affected SMC behavior: cells maintained their contractile phenotype and spread uniformly throughout the whole wall thickness. Conversely, static culture induced a noticeable polarization of cell distribution to the outer rim of the constructs and a sharp reduction in total cell density. Overall, coupling the use of a novel dual-mode bioreactor with engineered collagen-gel-based tubular constructs demonstrated to be an interesting technology to investigate the modulation of cell and tissue behavior under controlled mechanically conditioned in vitro maturation.

Engineering cells with intracellular agent–loaded microparticles to control cell phenotype

PubMed Central

Ankrum, James A; Miranda, Oscar R; Ng, Kelvin S; Sarkar, Debanjan; Xu, Chenjie; Karp, Jeffrey M

2014-01-01

Cell therapies enable unprecedented treatment options to replace tissues, destroy tumors and facilitate regeneration. The greatest challenge facing cell therapy is the inability to control the fate and function of cells after transplantation. We have developed an approach to control cell phenotype in vitro and after transplantation by engineering cells with intracellular depots that continuously release phenotype-altering agents for days to weeks. The platform enables control of cells’ secretome, viability, proliferation and differentiation, and the platform can be used to deliver drugs or other factors (e.g., dexamethasone, rhodamine and iron oxide) to the cell’s microenvironment. The preparation, efficient internalization and intracellular stabilization of ~1-μm drug-loaded microparticles are critical for establishing sustained control of cell phenotype. Herein we provide a protocol to generate and characterize micrometer-sized agent-doped poly(lactic-co-glycolic) acid (PLGA) particles by using a single-emulsion evaporation technique (7 h), to uniformly engineer cultured cells (15 h), to confirm particle internalization and to troubleshoot commonly experienced obstacles. PMID:24407352

Comparison of Phenotypic and Functional Characteristics Between Canine Non-B, Non-T Natural Killer Lymphocytes and CD3+CD5dimCD21- Cytotoxic Large Granular Lymphocytes.

PubMed

Lee, Soo-Hyeon; Shin, Dong-Jun; Kim, Yoseop; Kim, Cheol-Jung; Lee, Je-Jung; Yoon, Mee Sun; Uong, Tung Nguyen Thanh; Yu, Dohyeon; Jung, Ji-Youn; Cho, Duck; Jung, Bock-Gie; Kim, Sang-Ki; Suh, Guk-Hyun

2018-01-01

Natural killer (NK) cells play a pivotal role in the immune response against infections and malignant transformation, and adopted transfer of NK cells is thought to be a promising therapeutic approach for cancer patients. Previous reports describing the phenotypic features of canine NK cells have produced inconsistent results. Canine NK cells are still defined as non-B and non-T (CD3 - CD21 - ) large granular lymphocytes. However, a few reports have demonstrated that canine NK cells share the phenotypic characteristics of T lymphocytes, and that CD3 + CD5 dim CD21 - lymphocytes are putative canine NK cells. Based on our previous reports, we hypothesized that phenotypic modulation could occur between these two populations during activation. In this study, we investigated the phenotypic and functional differences between CD3 + CD5 dim CD21 - (cytotoxic large granular lymphocytes) and CD3 - CD5 - CD21 - NK lymphocytes before and after culture of peripheral blood mononuclear cells isolated from normal dogs. The results of this study show that CD3 + CD5 dim CD21 - lymphocytes can be differentiated into non-B, non-T NK (CD3 - CD5 - CD21 - TCRαβ - TCRγδ - GranzymeB + ) lymphocytes through phenotypic modulation in response to cytokine stimulation. In vitro studies of purified CD3 + CD5 dim CD21 - cells showed that CD3 - CD5 - CD21 - cells are derived from CD3 + CD5 dim CD21 - cells through phenotypic modulation. CD3 + CD5 dim CD21 - cells share more NK cell functional characteristics compared with CD3 - CD5 - CD21 - cells, including the expression of T-box transcription factors (Eomes, T-bet), the production of granzyme B and interferon-γ, and the expression of NK cell-related molecular receptors such as NKG2D and NKp30. In conclusion, the results of this study suggest that CD3 + CD5 dim CD21 - and CD3 - CD5 - CD21 - cells both contain a subset of putative NK cells, and the difference between the two populations may be due to the degree of maturation.

CXCR6, a newly defined biomarker of tissue-specific stem cell asymmetric self-renewal, identifies more aggressive human melanoma cancer stem cells.

PubMed

Taghizadeh, Rouzbeh; Noh, Minsoo; Huh, Yang Hoon; Ciusani, Emilio; Sigalotti, Luca; Maio, Michele; Arosio, Beatrice; Nicotra, Maria R; Natali, PierGiorgio; Sherley, James L; La Porta, Caterina A M

2010-12-22

A fundamental problem in cancer research is identifying the cell type that is capable of sustaining neoplastic growth and its origin from normal tissue cells. Recent investigations of a variety of tumor types have shown that phenotypically identifiable and isolable subfractions of cells possess the tumor-forming ability. In the present paper, using two lineage-related human melanoma cell lines, primary melanoma line IGR39 and its metastatic derivative line IGR37, two main observations are reported. The first one is the first phenotypic evidence to support the origin of melanoma cancer stem cells (CSCs) from mutated tissue-specific stem cells; and the second one is the identification of a more aggressive subpopulation of CSCs in melanoma that are CXCR6+. We defined CXCR6 as a new biomarker for tissue-specific stem cell asymmetric self-renewal. Thus, the relationship between melanoma formation and ABCG2 and CXCR6 expression was investigated. Consistent with their non-metastatic character, unsorted IGR39 cells formed significantly smaller tumors than unsorted IGR37 cells. In addition, ABCG2+ cells produced tumors that had a 2-fold greater mass than tumors produced by unsorted cells or ABCG2- cells. CXCR6+ cells produced more aggressive tumors. CXCR6 identifies a more discrete subpopulation of cultured human melanoma cells with a more aggressive MCSC phenotype than cells selected on the basis of the ABCG2+ phenotype alone. The association of a more aggressive tumor phenotype with asymmetric self-renewal phenotype reveals a previously unrecognized aspect of tumor cell physiology. Namely, the retention of some tissue-specific stem cell attributes, like the ability to asymmetrically self-renew, impacts the natural history of human tumor development. Knowledge of this new aspect of tumor development and progression may provide new targets for cancer prevention and treatment.

Osteoblast Specific Overexpression of Human Interleukin-7 Rescues the Bone Mass Phenotype of Interleukin-7 Deficient Female Mice

PubMed Central

Aguila, Hector L.; Mun, Se Hwan; Kalinowski, Judith; Adams, Douglas J.; Lorenzo, Joseph A.; Lee, Sun-Kyeong

2012-01-01

Interleukin-7 is a critical cytokine for lymphoid development and a direct inhibitor of in vitro osteoclastogenesis in murine bone marrow cultures. To explore the role of IL-7 in bone, we generated transgenic mouse lines bearing the 2.3 Kb rat collagen 1A1 promoter driving the expression of human IL-7 specifically in osteoblasts. In addition we crossed these mice with IL-7 deficient mice to determine if the alterations in lymphopoiesis, bone mass and osteoclast formation observed in the IL-7 KO mice could be rescued by osteoblast-specific overexpression of IL-7. Here we show that mice overexpressing human IL-7 in the osteoblast lineage demonstrated increased trabecular bone volume in vivo by µCT and decreased osteoclast formation in vitro. Furthermore, targeted overexpression of IL-7 in osteoblasts rescued the osteopenic bone phenotype and B cell development of IL-7 KO mice but did not have an effect on T lymphopoiesis, which occurs in the periphery. The bone phenotypes in IL-7 KO mice and targeted IL-7 overexpressing mouse models were observed only in females. These results likely reflect both a direct inhibitory effects of IL-7 on osteoclastogenesis in vivo and gender specific differences in responses to IL-7. PMID:22258693

Merkel Cell-Driven BDNF Signaling Specifies SAI Neuron Molecular and Electrophysiological Phenotypes.

PubMed

Reed-Geaghan, Erin G; Wright, Margaret C; See, Lauren A; Adelman, Peter C; Lee, Kuan Hsien; Koerber, H Richard; Maricich, Stephen M

2016-04-13

The extent to which the skin instructs peripheral somatosensory neuron maturation is unknown. We studied this question in Merkel cell-neurite complexes, where slowly adapting type I (SAI) neurons innervate skin-derived Merkel cells. Transgenic mice lacking Merkel cells had normal dorsal root ganglion (DRG) neuron numbers, but fewer DRG neurons expressed the SAI markers TrkB, TrkC, and Ret. Merkel cell ablation also decreased downstream TrkB signaling in DRGs, and altered the expression of genes associated with SAI development and function. Skin- and Merkel cell-specific deletion of Bdnf during embryogenesis, but not postnatal Bdnf deletion or Ntf3 deletion, reproduced these results. Furthermore, prototypical SAI electrophysiological signatures were absent from skin regions where Bdnf was deleted in embryonic Merkel cells. We conclude that BDNF produced by Merkel cells during a precise embryonic period guides SAI neuron development, providing the first direct evidence that the skin instructs sensory neuron molecular and functional maturation. Peripheral sensory neurons show incredible phenotypic and functional diversity that is initiated early by cell-autonomous and local environmental factors found within the DRG. However, the contribution of target tissues to subsequent sensory neuron development remains unknown. We show that Merkel cells are required for the molecular and functional maturation of the SAI neurons that innervate them. We also show that this process is controlled by BDNF signaling. These findings provide new insights into the regulation of somatosensory neuron development and reveal a novel way in which Merkel cells participate in mechanosensation. Copyright © 2016 the authors 0270-6474/16/364362-15$15.00/0.

Dynamic equilibrium of heterogeneous and interconvertible multipotent hematopoietic cell subsets

PubMed Central

Weston, Wendy; Zayas, Jennifer; Perez, Ruben; George, John; Jurecic, Roland

2014-01-01

Populations of hematopoietic stem cells and progenitors are quite heterogeneous and consist of multiple cell subsets with distinct phenotypic and functional characteristics. Some of these subsets also appear to be interconvertible and oscillate between functionally distinct states. The multipotent hematopoietic cell line EML has emerged as a unique model to study the heterogeneity and interconvertibility of multipotent hematopoietic cells. Here we describe extensive phenotypic and functional heterogeneity of EML cells which stems from the coexistence of multiple cell subsets. Each of these subsets is phenotypically and functionally heterogeneous, and displays distinct multilineage differentiation potential, cell cycle profile, proliferation kinetics, and expression pattern of HSC markers and some of the key lineage-associated transcription factors. Analysis of their maintenance revealed that on a population level all EML cell subsets exhibit cell-autonomous interconvertible properties, with the capacity to generate all other subsets and re-establish complete parental EML cell population. Moreover, all EML cell subsets generated during multiple cell generations maintain their distinct phenotypic and functional signatures and interconvertible properties. The model of EML cell line suggests that interconvertible multipotent hematopoietic cell subsets coexist in a homeostatically maintained dynamic equilibrium which is regulated by currently unknown cell-intrinsic mechanisms. PMID:24903657

Dynamic equilibrium of heterogeneous and interconvertible multipotent hematopoietic cell subsets.

PubMed

Weston, Wendy; Zayas, Jennifer; Perez, Ruben; George, John; Jurecic, Roland

2014-06-06

Populations of hematopoietic stem cells and progenitors are quite heterogeneous and consist of multiple cell subsets with distinct phenotypic and functional characteristics. Some of these subsets also appear to be interconvertible and oscillate between functionally distinct states. The multipotent hematopoietic cell line EML has emerged as a unique model to study the heterogeneity and interconvertibility of multipotent hematopoietic cells. Here we describe extensive phenotypic and functional heterogeneity of EML cells which stems from the coexistence of multiple cell subsets. Each of these subsets is phenotypically and functionally heterogeneous, and displays distinct multilineage differentiation potential, cell cycle profile, proliferation kinetics, and expression pattern of HSC markers and some of the key lineage-associated transcription factors. Analysis of their maintenance revealed that on a population level all EML cell subsets exhibit cell-autonomous interconvertible properties, with the capacity to generate all other subsets and re-establish complete parental EML cell population. Moreover, all EML cell subsets generated during multiple cell generations maintain their distinct phenotypic and functional signatures and interconvertible properties. The model of EML cell line suggests that interconvertible multipotent hematopoietic cell subsets coexist in a homeostatically maintained dynamic equilibrium which is regulated by currently unknown cell-intrinsic mechanisms.

Cryopreservation of human vascular umbilical cord cells under good manufacturing practice conditions for future cell banks

PubMed Central

2012-01-01

Background In vitro fabricated tissue engineered vascular constructs could provide an alternative to conventional substitutes. A crucial factor for tissue engineering of vascular constructs is an appropriate cell source. Vascular cells from the human umbilical cord can be directly isolated and cryopreserved until needed. Currently no cell bank for human vascular cells is available. Therefore, the establishment of a future human vascular cell bank conforming to good manufacturing practice (GMP) conditions is desirable for therapeutic applications such as tissue engineered cardiovascular constructs. Materials and methods A fundamental step was the adaption of conventional research and development starting materials to GMP compliant starting materials. Human umbilical cord artery derived cells (HUCAC) and human umbilical vein endothelial cells (HUVEC) were isolated, cultivated, cryopreserved (short- and long-term) directly after primary culture and recultivated subsequently. Cell viability, expression of cellular markers and proliferation potential of fresh and cryopreserved cells were studied using trypan blue staining, flow cytometry analysis, immunofluorescence staining and proliferation assays. Statistical analyses were performed using Student’s t-test. Results Sufficient numbers of isolated cells with acceptable viabilities and homogenous expression of cellular markers confirmed that the isolation procedure was successful using GMP compliant starting materials. The influence of cryopreservation was marginal, because cryopreserved cells mostly maintain phenotypic and functional characteristics similar to those of fresh cells. Phenotypic studies revealed that fresh cultivated and cryopreserved HUCAC were positive for alpha smooth muscle actin, CD90, CD105, CD73, CD29, CD44, CD166 and negative for smoothelin. HUVEC expressed CD31, CD146, CD105 and CD144 but not alpha smooth muscle actin. Functional analysis demonstrated acceptable viability and sufficient proliferation properties of cryopreserved HUCAC and HUVEC. Conclusion Adaptation of cell isolation, cultivation and cryopreservation to GMP compliant starting materials was successful. Cryopreservation did not influence cell properties with lasting impact, confirming that the application of vascular cells from the human umbilical cord is feasible for cell banking. A specific cellular marker expression profile was established for HUCAC and HUVEC using flow cytometry analysis, applicable as a GMP compliant quality control. Use of these cells for the future fabrication of advanced therapy medicinal products GMP conditions are required by the regulatory authority. PMID:22591741

KLF4-dependent epigenetic remodeling modulates podocyte phenotypes and attenuates proteinuria

PubMed Central

Hayashi, Kaori; Sasamura, Hiroyuki; Nakamura, Mari; Azegami, Tatsuhiko; Oguchi, Hideyo; Sakamaki, Yusuke; Itoh, Hiroshi

2014-01-01

The transcription factor Kruppel-like factor 4 (KLF4) has the ability, along with other factors, to reprogram somatic cells into induced pluripotent stem (iPS) cells. Here, we determined that KLF4 is expressed in kidney glomerular podocytes and is decreased in both animal models and humans exhibiting a proteinuric. Transient restoration of KLF4 expression in podocytes of diseased glomeruli in vivo, either by gene transfer or transgenic expression, resulted in a sustained increase in nephrin expression and a decrease in albuminuria. In mice harboring podocyte-specific deletion of Klf4, adriamycin-induced proteinuria was substantially exacerbated, although these animals displayed minimal phenotypical changes prior to adriamycin administration. KLF4 overexpression in cultured human podocytes increased expression of nephrin and other epithelial markers and reduced mesenchymal gene expression. DNA methylation profiling and bisulfite genomic sequencing revealed that KLF4 expression reduced methylation at the nephrin promoter and the promoters of other epithelial markers; however, methylation was increased at the promoters of genes encoding mesenchymal markers, suggesting selective epigenetic regulation of podocyte gene expression. Together, these results suggest that KLF4 epigenetically modulates podocyte phenotype and function and that the podocyte epigenome can be targeted for direct intervention and reduction of proteinuria. PMID:24812666

miR-181d/MALT1 regulatory axis attenuates mesenchymal phenotype through NF-κB pathways in glioblastoma.

PubMed

Yang, Fan; Liu, Xing; Liu, Yanwei; Liu, Yuqing; Zhang, Chuanbao; Wang, Zheng; Jiang, Tao; Wang, Yongzhi

2017-06-28

The mesenchymal (MES) subtype of glioblastoma (GBM) indicated a more malignant phenotype and worse prognosis compared with their proneural (PN) counterpart. The plasticity between PN and MES transcriptome signatures provided an approach for clinical intervention. However, few miRNAs have been identified to participate in the shift between subtypes. Here, we utilized transcriptomic data and experimental evidences to prove that miR-181d was a novel regulator of NFκB signaling pathway by directly repressing MALT1, leading to induced PN markers and reduced MES genes. Functionally, ectopic expression of miR-181d suppressed GBM cell proliferation, colony formation and anchor-independent growth, as well as migration, invasion and tube formation. Moreover, miR-181d overexpression increased radio- and chemo-sensitivity for GBM cells. Rescue of MALT1 could partially reverse the effects of miR-181d in GBM malignant behaviors. Clinically, miR-181d could serve as a prognostic indicator for GBM patients. Taken together, we concluded that loss of miR-181d contributes to aggressive biological processes associated with MES phenotype via NFκB signaling, which broaden our insights into the underlying mechanisms in subtype transition and miRNA-based tailored medicine for GBM management. Copyright © 2017 Elsevier B.V. All rights reserved.

Stem cell-derived neurons in the development of targeted treatment for schizophrenia and bipolar disorder.

PubMed

Watmuff, Bradley; Liu, Bangyan; Karmacharya, Rakesh

2017-04-01

The recent advent of induced pluripotent stem cells has enabled the study of patient-specific and disease-related neurons in vitro and has facilitated new directions of inquiry into disease mechanisms. With these approaches, we now have the possibility of correlating ex vivo cellular phenotypes with individual patient response to treatment and/or side effects, which makes targeted treatments for schizophrenia and bipolar disorder a distinct prospect in the coming years. Here, we briefly review the current state of stem cell-based models and explore studies that are providing new insights into the disease biology of schizophrenia and bipolar disorder, which are laying the foundations for the development of novel targeted therapies.

Interleukin-17A and vascular remodelling in severe asthma; lack of evidence for a direct role.

PubMed

Panariti, A; Baglole, C J; Sanchez, V; Eidelman, D H; Hussain, S; Olivenstein, R; Martin, J G; Hamid, Q

2018-04-01

Bronchial vascular remodelling may contribute to the severity of airway narrowing through mucosal congestion. Interleukin (IL)-17A is associated with the most severe asthmatic phenotype but whether it might contribute to vascular remodelling is uncertain. To assess vascular remodelling in severe asthma and whether IL-17A directly or indirectly may cause endothelial cell activation and angiogenesis. Bronchial vascularization was quantified in asthmatic subjects, COPD and healthy subjects together with the number of IL-17A + cells as well as the concentration of angiogenic factors in the sputum. The effect of IL-17A on in vitro angiogenesis, cell migration and endothelial permeability was assessed directly on primary human lung microvascular endothelial cells (HMVEC-L) or indirectly with conditioned medium derived from normal bronchial epithelial cells (NHBEC), fibroblasts (NHBF) and airway smooth muscle cells (ASMC) after IL-17A stimulation. Severe asthmatics have increased vascularity compared to the other groups, which correlates positively with the concentrations of angiogenic factors in sputum. Interestingly, we demonstrated that increased bronchial vascularity correlates positively with the number of subepithelial IL-17A + cells. However IL-17A had no direct effect on HMVEC-L function but it enhanced endothelial tube formation and cell migration through the production of angiogenic factors by NHBE and ASMC. Our results shed light on the role of IL-17A in vascular remodelling, most likely through stimulating the synthesis of other angiogenic factors. Knowledge of these pathways may aid in the identification of new therapeutic targets. © 2018 John Wiley & Sons Ltd.

Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures

PubMed Central

Fluri, David A.; Tonge, Peter D.; Song, Hannah; Baptista, Ricardo P.; Shakiba, Nika; Shukla, Shreya; Clarke, Geoffrey; Nagy, Andras; Zandstra, Peter W.

2016-01-01

We demonstrate derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum- and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension (S) reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor expressing cells based on their differential survival and proliferation in suspension. Seamless integration of SiPSC reprogramming and directed differentiation enabled the scalable production of functionally and phenotypically defined cardiac cells in a continuous single cell- and small aggregate-based process. This method is an important step towards the development of a robust PSC generation, expansion and differentiation technology. PMID:22447133

Virus-induced gene silencing offers a functional genomics platform for studying plant cell wall formation.

PubMed

Zhu, Xiaohong; Pattathil, Sivakumar; Mazumder, Koushik; Brehm, Amanda; Hahn, Michael G; Dinesh-Kumar, S P; Joshi, Chandrashekhar P

2010-09-01

Virus-induced gene silencing (VIGS) is a powerful genetic tool for rapid assessment of plant gene functions in the post-genomic era. Here, we successfully implemented a Tobacco Rattle Virus (TRV)-based VIGS system to study functions of genes involved in either primary or secondary cell wall formation in Nicotiana benthamiana plants. A 3-week post-VIGS time frame is sufficient to observe phenotypic alterations in the anatomical structure of stems and chemical composition of the primary and secondary cell walls. We used cell wall glycan-directed monoclonal antibodies to demonstrate that alteration of cell wall polymer synthesis during the secondary growth phase of VIGS plants has profound effects on the extractability of components from woody stem cell walls. Therefore, TRV-based VIGS together with cell wall component profiling methods provide a high-throughput gene discovery platform for studying plant cell wall formation from a bioenergy perspective.

Social parasitism and the molecular basis of phenotypic evolution.

PubMed

Cini, Alessandro; Patalano, Solenn; Segonds-Pichon, Anne; Busby, George B J; Cervo, Rita; Sumner, Seirian

2015-01-01

Contrasting phenotypes arise from similar genomes through a combination of losses, gains, co-option and modifications of inherited genomic material. Understanding the molecular basis of this phenotypic diversity is a fundamental challenge in modern evolutionary biology. Comparisons of the genes and their expression patterns underlying traits in closely related species offer an unrivaled opportunity to evaluate the extent to which genomic material is reorganized to produce novel traits. Advances in molecular methods now allow us to dissect the molecular machinery underlying phenotypic diversity in almost any organism, from single-celled entities to the most complex vertebrates. Here we discuss how comparisons of social parasites and their free-living hosts may provide unique insights into the molecular basis of phenotypic evolution. Social parasites evolve from a eusocial ancestor and are specialized to exploit the socially acquired resources of their closely-related eusocial host. Molecular comparisons of such species pairs can reveal how genomic material is re-organized in the loss of ancestral traits (i.e., of free-living traits in the parasites) and the gain of new ones (i.e., specialist traits required for a parasitic lifestyle). We define hypotheses on the molecular basis of phenotypes in the evolution of social parasitism and discuss their wider application in our understanding of the molecular basis of phenotypic diversity within the theoretical framework of phenotypic plasticity and shifting reaction norms. Currently there are no data available to test these hypotheses, and so we also provide some proof of concept data using the paper wasp social parasite/host system (Polistes sulcifer-Polistes dominula). This conceptual framework and first empirical data provide a spring-board for directing future genomic analyses on exploiting social parasites as a route to understanding the evolution of phenotypic specialization.

Social parasitism and the molecular basis of phenotypic evolution

PubMed Central

Cini, Alessandro; Patalano, Solenn; Segonds-Pichon, Anne; Busby, George B. J.; Cervo, Rita; Sumner, Seirian

2015-01-01

Contrasting phenotypes arise from similar genomes through a combination of losses, gains, co-option and modifications of inherited genomic material. Understanding the molecular basis of this phenotypic diversity is a fundamental challenge in modern evolutionary biology. Comparisons of the genes and their expression patterns underlying traits in closely related species offer an unrivaled opportunity to evaluate the extent to which genomic material is reorganized to produce novel traits. Advances in molecular methods now allow us to dissect the molecular machinery underlying phenotypic diversity in almost any organism, from single-celled entities to the most complex vertebrates. Here we discuss how comparisons of social parasites and their free-living hosts may provide unique insights into the molecular basis of phenotypic evolution. Social parasites evolve from a eusocial ancestor and are specialized to exploit the socially acquired resources of their closely-related eusocial host. Molecular comparisons of such species pairs can reveal how genomic material is re-organized in the loss of ancestral traits (i.e., of free-living traits in the parasites) and the gain of new ones (i.e., specialist traits required for a parasitic lifestyle). We define hypotheses on the molecular basis of phenotypes in the evolution of social parasitism and discuss their wider application in our understanding of the molecular basis of phenotypic diversity within the theoretical framework of phenotypic plasticity and shifting reaction norms. Currently there are no data available to test these hypotheses, and so we also provide some proof of concept data using the paper wasp social parasite/host system (Polistes sulcifer—Polistes dominula). This conceptual framework and first empirical data provide a spring-board for directing future genomic analyses on exploiting social parasites as a route to understanding the evolution of phenotypic specialization. PMID:25741361

Bone Marrow Stromal Cells Contribute to Bone Formation Following Infusion into Femoral Cavities of a Mouse Model of Osteogenesis Imperfecta

PubMed Central

Li, Feng; Wang, Xujun; Niyibizi, Christopher

2010-01-01

Currently, there are conflicting data in literature regarding contribution of bone marrow stromal cells (BMSCs) to bone formation when the cells are systemically delivered in recipient animals. To understand if BMSCs contribute to bone cell phenotype and bone formation in osteogenesis imperfecta bones (OI), MSCs marked with GFP were directly infused into the femurs of a mouse model of OI (oim). The contribution of the cells to the cell phenotype and bone formation was assessed by histology, immunohistochemistry and biomechanical loading of recipient bones. Two weeks following infusion of BMSCs, histological examination of the recipient femurs demonstrated presence of new bone when compared to femurs injected with saline which showed little or no bone formation. The new bone contained few donor cells as demonstrated by GFP fluorescence. At six weeks following cell injection, new bone was still detectable in the recipient femurs but was enhanced by injection of the cells suspended in pepsin solublized type I collagen. Immunofluorescence and immunohistochemical staining showed that donor GFP positive cells in the new bone were localized with osteocalcin expressing cells suggesting that the cells differentiated into osteoblasts in vivo. Biomechanical loading to failure in thee point bending, revealed that, femurs infused with BMSCs in PBS or in soluble type I collagen were biomechanically stronger than those injected with PBS or type I collagen alone. Taken together, the results indicate that transplanted cells differentiated into osteoblasts in vivo and contributed to bone formation in vivo; we also speculate that donor cells induced differentiation or recruitment of endogenous cells to initiate reparative process at early stages following transplantation. PMID:20570757

Delineation of a novel pre-B cell component in plasma cell myeloma: immunochemical, immunophenotypic, genotypic, cytologic, cell culture, and kinetic features.

PubMed

Grogan, T M; Durie, B G; Lomen, C; Spier, C; Wirt, D P; Nagle, R; Wilson, G S; Richter, L; Vela, E; Maxey, V

1987-10-01

A novel pre-B cell component in direct and cultured myeloma bone marrow material has been delineated by using immunochemistry and flow cytometry techniques. Our phenotypic studies suggest a novel hybrid expression of pre-B and plasma cell antigens with coexpression of cytoplasmic mu, common acute lymphoblastic leukemia antigen, terminal deoxynucleotidyl transferase, and plasma cell antigens (PCA-1 and PC-1). This suggests that myeloma pre-B-like cells are aberrant malignant cells and not normal pre-B lymphocytic counterparts. With the advantage of a pure and stable source of these cells from M3 culture to allow molecular characterization, we performed one- and two-dimensional gel electrophoresis and Western blotting. We found that the cytoplasmic mu in myeloma pre-B-like cells has a molecular weight of 74,000 daltons and an isoelectric point of 6.3 and that it is strikingly homogeneous and discrete in size and charge compared with standard secretory mu, which suggests an aberrant, mutant, or monoclonal form of mu. Monoclonality was further evidenced by heavy- and light-chain immunoglobulin gene rearrangements demonstrated with JH and C kappa probes. We also established that this novel myeloma pre-B component is a major proliferative element as determined by double-labeling experiments with phenotype coupled to labeling/proliferative indexes. Our stimulatory studies indicate some capacity of these cells to mature on exposure to phorbol esters. These myeloma pre-B cells may represent the stem cell or self-renewal component in myeloma. Our establishment of these cells in long-term culture offers a considerable asset in studying the immature cells, which may be critical to the immortalization of myeloma.

FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells.

PubMed

Lee, Hyung-Ok; Mullins, Stefanie R; Franco-Barraza, Janusz; Valianou, Matthildi; Cukierman, Edna; Cheng, Jonathan D

2011-06-13

Alterations towards a permissive stromal microenvironment provide important cues for tumor growth, invasion, and metastasis. In this study, Fibroblast activation protein (FAP), a serine protease selectively produced by tumor-associated fibroblasts in over 90% of epithelial tumors, was used as a platform for studying tumor-stromal interactions. We tested the hypothesis that FAP enzymatic activity locally modifies stromal ECM (extracellular matrix) components thus facilitating the formation of a permissive microenvironment promoting tumor invasion in human pancreatic cancer. We generated a tetracycline-inducible FAP overexpressing fibroblastic cell line to synthesize an in vivo-like 3-dimensional (3D) matrix system which was utilized as a stromal landscape for studying matrix-induced cancer cell behaviors. A FAP-dependent topographical and compositional alteration of the ECM was characterized by measuring the relative orientation angles of fibronectin fibers and by Western blot analyses. The role of FAP in the matrix-induced permissive tumor behavior was assessed in Panc-1 cells in assorted matrices by time-lapse acquisition assays. Also, FAP+ matrix-induced regulatory molecules in cancer cells were determined by Western blot analyses. We observed that FAP remodels the ECM through modulating protein levels, as well as through increasing levels of fibronectin and collagen fiber organization. FAP-dependent architectural/compositional alterations of the ECM promote tumor invasion along characteristic parallel fiber orientations, as demonstrated by enhanced directionality and velocity of pancreatic cancer cells on FAP+ matrices. This phenotype can be reversed by inhibition of FAP enzymatic activity during matrix production resulting in the disorganization of the ECM and impeded tumor invasion. We also report that the FAP+ matrix-induced tumor invasion phenotype is β1-integrin/FAK mediated. Cancer cell invasiveness can be affected by alterations in the tumor microenvironment. Disruption of FAP activity and β1-integrins may abrogate the invasive capabilities of pancreatic and other tumors by disrupting the FAP-directed organization of stromal ECM and blocking β1-integrin dependent cell-matrix interactions. This provides a novel preclinical rationale for therapeutics aimed at interfering with the architectural organization of tumor-associated ECM. Better understanding of the stromal influences that fuel progressive tumorigenic behaviors may allow the effective future use of targeted therapeutics aimed at disrupting specific tumor-stromal interactions. © 2011 Lee et al; licensee BioMed Central Ltd.

FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells

PubMed Central

2011-01-01

Background Alterations towards a permissive stromal microenvironment provide important cues for tumor growth, invasion, and metastasis. In this study, Fibroblast activation protein (FAP), a serine protease selectively produced by tumor-associated fibroblasts in over 90% of epithelial tumors, was used as a platform for studying tumor-stromal interactions. We tested the hypothesis that FAP enzymatic activity locally modifies stromal ECM (extracellular matrix) components thus facilitating the formation of a permissive microenvironment promoting tumor invasion in human pancreatic cancer. Methods We generated a tetracycline-inducible FAP overexpressing fibroblastic cell line to synthesize an in vivo-like 3-dimensional (3D) matrix system which was utilized as a stromal landscape for studying matrix-induced cancer cell behaviors. A FAP-dependent topographical and compositional alteration of the ECM was characterized by measuring the relative orientation angles of fibronectin fibers and by Western blot analyses. The role of FAP in the matrix-induced permissive tumor behavior was assessed in Panc-1 cells in assorted matrices by time-lapse acquisition assays. Also, FAP+ matrix-induced regulatory molecules in cancer cells were determined by Western blot analyses. Results We observed that FAP remodels the ECM through modulating protein levels, as well as through increasing levels of fibronectin and collagen fiber organization. FAP-dependent architectural/compositional alterations of the ECM promote tumor invasion along characteristic parallel fiber orientations, as demonstrated by enhanced directionality and velocity of pancreatic cancer cells on FAP+ matrices. This phenotype can be reversed by inhibition of FAP enzymatic activity during matrix production resulting in the disorganization of the ECM and impeded tumor invasion. We also report that the FAP+ matrix-induced tumor invasion phenotype is β1-integrin/FAK mediated. Conclusion Cancer cell invasiveness can be affected by alterations in the tumor microenvironment. Disruption of FAP activity and β1-integrins may abrogate the invasive capabilities of pancreatic and other tumors by disrupting the FAP-directed organization of stromal ECM and blocking β1-integrin dependent cell-matrix interactions. This provides a novel preclinical rationale for therapeutics aimed at interfering with the architectural organization of tumor-associated ECM. Better understanding of the stromal influences that fuel progressive tumorigenic behaviors may allow the effective future use of targeted therapeutics aimed at disrupting specific tumor-stromal interactions. PMID:21668992

A systems biology approach to defining regulatory mechanisms for cartilage and tendon cell phenotypes.

PubMed

Mueller, A J; Tew, S R; Vasieva, O; Clegg, P D; Canty-Laird, E G

2016-09-27

Phenotypic plasticity of adult somatic cells has provided emerging avenues for the development of regenerative therapeutics. In musculoskeletal biology the mechanistic regulatory networks of genes governing the phenotypic plasticity of cartilage and tendon cells has not been considered systematically. Additionally, a lack of strategies to effectively reproduce in vitro functional models of cartilage and tendon is retarding progress in this field. De- and redifferentiation represent phenotypic transitions that may contribute to loss of function in ageing musculoskeletal tissues. Applying a systems biology network analysis approach to global gene expression profiles derived from common in vitro culture systems (monolayer and three-dimensional cultures) this study demonstrates common regulatory mechanisms governing de- and redifferentiation transitions in cartilage and tendon cells. Furthermore, evidence of convergence of gene expression profiles during monolayer expansion of cartilage and tendon cells, and the expression of key developmental markers, challenges the physiological relevance of this culture system. The study also suggests that oxidative stress and PI3K signalling pathways are key modulators of in vitro phenotypes for cells of musculoskeletal origin.

Stochastic modeling and experimental analysis of phenotypic switching and survival of cancer cells under stress

NASA Astrophysics Data System (ADS)

Zamani Dahaj, Seyed Alireza; Kumar, Niraj; Sundaram, Bala; Celli, Jonathan; Kulkarni, Rahul

The phenotypic heterogeneity of cancer cells is critical to their survival under stress. A significant contribution to heterogeneity of cancer calls derives from the epithelial-mesenchymal transition (EMT), a conserved cellular program that is crucial for embryonic development. Several studies have investigated the role of EMT in growth of early stage tumors into invasive malignancies. Also, EMT has been closely associated with the acquisition of chemoresistance properties in cancer cells. Motivated by these studies, we analyze multi-phenotype stochastic models of the evolution of cancers cell populations under stress. We derive analytical results for time-dependent probability distributions that provide insights into the competing rates underlying phenotypic switching (e.g. during EMT) and the corresponding survival of cancer cells. Experimentally, we evaluate these model-based predictions by imaging human pancreatic cancer cell lines grown with and without cytotoxic agents and measure growth kinetics, survival, morphological changes and (terminal evaluation of) biomarkers with associated epithelial and mesenchymal phenotypes. The results derived suggest approaches for distinguishing between adaptation and selection scenarios for survival in the presence of external stresses.

A Computational Study of Phenotype Switching in Bacillus Subtilis Biofilm

NASA Astrophysics Data System (ADS)

Smith, Howard; Wang, Xiaoling; Jiang, Yi

Bacillus Subtilis (B. Subtilis), is known to differentiate into three main phenotypes during biofilm growth. Novel techniques to track the spatial and temporal evolution of the three main phenotypes exhibited by B. Subtilis have been developed. However, the techniques do not explain the environmental causes of the phenotype switching and how this leads to the spatiotemporal organization of the biofilm. We hypothesize that cells switch their phenotype according to nutrients and autoinducer levels. We test the hypothesis using a hybrid agent-based and continuous model. The bacteria in our model are individual cells that can (i) grow and divide by the intake of nutrients, (ii) produce and secrete EPS, (iii) form spores and (iv) produce an auto inducer. Using a threshold for nutrient and thresholds for autoinducers, we were able to reproduce the experimental spatiotemporal dynamics. From our simulations we observed that in order to reproduce experimental results, two different autoinducers were necessary. The results also suggest that low-EPS producing biofilms generally obtained higher cell populations. Furthermore, most of the cells that become spore forming cells arise from matrix producing cells.

Loss of end-differentiated β-cell phenotype following pancreatic islet transplantation.

PubMed

Anderson, S J; White, M G; Armour, S L; Maheshwari, R; Tiniakos, D; Muller, Y D; Berishvili, E; Berney, T; Shaw, J A M

2018-03-01

Replacement of pancreatic β-cells through deceased donor islet transplantation is a proven therapy for preventing recurrent life-threatening hypoglycemia in type 1 diabetes. Although near-normal glucose levels and insulin independence can be maintained for many years following successful islet transplantation, restoration of normal functional β-cell mass has remained elusive. It has recently been proposed that dedifferentiation/plasticity towards other endocrine phenotypes may play an important role in stress-induced β-cell dysfunction in type 2 diabetes. Here we report loss of end-differentiated β-cell phenotype in 2 intraportal islet allotransplant recipients. Despite excellent graft function and sustained insulin independence, all examined insulin-positive cells had lost expression of the end-differentiation marker, urocortin-3, or appeared to co-express the α-cell marker, glucagon. In contrast, no insulin + /urocortin-3 - cells were seen in nondiabetic deceased donor control pancreatic islets. Loss of end-differentiated phenotype may facilitate β-cell survival during the stresses associated with islet isolation and culture, in addition to sustained hypoxia following engraftment. As further refinements in islet isolation and culture are made in parallel with exploration of alternative β-cell sources, graft sites, and ultimately fully vascularized bioengineered insulin-secreting microtissues, differentiation status immunostaining provides a novel tool to assess whether fully mature β-cell phenotype has been maintained. © 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.

Calcium-mediated shaping of naive CD4 T-cell phenotype and function

PubMed Central

Guichard, Vincent; Bonilla, Nelly; Durand, Aurélie; Audemard-Verger, Alexandra; Guilbert, Thomas; Martin, Bruno

2017-01-01

Continuous contact with self-major histocompatibility complex ligands is essential for the survival of naive CD4 T cells. We have previously shown that the resulting tonic TCR signaling also influences their fate upon activation by increasing their ability to differentiate into induced/peripheral regulatory T cells. To decipher the molecular mechanisms governing this process, we here focus on the TCR signaling cascade and demonstrate that a rise in intracellular calcium levels is sufficient to modulate the phenotype of mouse naive CD4 T cells and to increase their sensitivity to regulatory T-cell polarization signals, both processes relying on calcineurin activation. Accordingly, in vivo calcineurin inhibition leads the most self-reactive naive CD4 T cells to adopt the phenotype of their less self-reactive cell-counterparts. Collectively, our findings demonstrate that calcium-mediated activation of the calcineurin pathway acts as a rheostat to shape both the phenotype and effector potential of naive CD4 T cells in the steady-state. PMID:29239722

CD147 (Basigin/Emmprin) identifies FoxP3+CD45RO+CTLA4+-activated human regulatory T cells.

PubMed

Solstad, Therese; Bains, Simer Jit; Landskron, Johannes; Aandahl, Einar Martin; Thiede, Bernd; Taskén, Kjetil; Torgersen, Knut Martin

2011-11-10

Human CD4(+)FoxP3(+) T cells are functionally and phenotypically heterogeneous providing plasticity to immune activation and regulation. To better understand the functional dynamics within this subset, we first used a combined strategy of subcellular fractionation and proteomics to describe differences at the protein level between highly purified human CD4(+)CD25(+) and CD4(+)CD25(-) T-cell populations. This identified a set of membrane proteins highly expressed on the cell surface of human regulatory T cells (Tregs), including CD71, CD95, CD147, and CD148. CD147 (Basigin or Emmprin) divided CD4(+)CD25(+) cells into distinct subsets. Furthermore, CD147, CD25, FoxP3, and in particular CTLA-4 expression correlated. Phenotypical and functional analyses suggested that CD147 marks the switch between resting (CD45RA(+)) and activated (CD45RO(+)) subsets within the FoxP3(+) T-cell population. Sorting of regulatory T cells into CD147(-) and CD147(+) populations demonstrated that CD147 identifies an activated and highly suppressive CD45RO(+) Treg subset. When analyzing CD4(+) T cells for their cytokine producing potential, CD147 levels grouped the FoxP3(+) subset into 3 categories with different ability to produce IL-2, TNF-α, IFN-γ, and IL-17. Together, this suggests that CD147 is a direct marker for activated Tregs within the CD4(+)FoxP3(+) subset and may provide means to manipulate cells important for immune homeostasis.

Indoxyl Sulfate Induces Apoptosis and Hypertrophy in Human Kidney Proximal Tubular Cells.

PubMed

Ellis, Robert J; Small, David M; Ng, Keng Lim; Vesey, David A; Vitetta, Luis; Francis, Ross S; Gobe, Glenda C; Morais, Christudas

2018-06-01

Indoxyl sulfate (IS) is a protein-bound uremic toxin that accumulates in patients with declining kidney function. Although generally thought of as a consequence of declining kidney function, emerging evidence demonstrates direct cytotoxic role of IS on endothelial cells and cardiomyocytes, largely through the expression of pro-inflammatory and pro-fibrotic factors. The direct toxicity of IS on human kidney proximal tubular epithelial cells (PTECs) remains a matter of debate. The current study explored the effect of IS on primary cultures of human PTECs and HK-2, an immortalized human PTEC line. Pathologically relevant concentrations of IS induced apoptosis and increased the expression of the proapoptotic molecule Bax in both cell types. IS impaired mitochondrial metabolic activity and induced cellular hypertrophy. Furthermore, statistically significant upregulation of pro-fibrotic (transforming growth factor-β, fibronectin) and pro-inflammatory molecules (interleukin-6, interleukin-8, and tumor necrosis factor-α) in response to IS was observed. Albumin had no influence on the toxicity of IS. The results of this study suggest that IS directly induced a pro-inflammatory and pro-fibrotic phenotype in proximal tubular cells. In light of the associated apoptosis, hypertrophy, and metabolic dysfunction, this study demonstrates that IS may play a role in the progression of chronic kidney disease.

Alternative dominance of the parental genomes in hybrid cells generated through the fusion of mouse embryonic stem cells with fibroblasts.

PubMed

Matveeva, Natalia M; Fishman, Veniamin S; Zakharova, Irina S; Shevchenko, Alexander I; Pristyazhnyuk, Inna E; Menzorov, Aleksei G; Serov, Oleg L

2017-12-22

For the first time, two types of hybrid cells with embryonic stem (ES) cell-like and fibroblast-like phenotypes were produced through the fusion of mouse ES cells with fibroblasts. Transcriptome analysis of 2,848 genes differentially expressed in the parental cells demonstrated that 34-43% of these genes are expressed in hybrid cells, consistent with their phenotypes; 25-29% of these genes display intermediate levels of expression, and 12-16% of these genes maintained expression at the parental cell level, inconsistent with the phenotype of the hybrid cell. Approximately 20% of the analyzed genes displayed unexpected expression patterns that differ from both parents. An unusual phenomenon was observed, namely, the illegitimate activation of Xist expression and the inactivation of one of two X-chromosomes in the near-tetraploid fibroblast-like hybrid cells, whereas both Xs were active before and after in vitro differentiation of the ES cell-like hybrid cells. These results and previous data obtained on heterokaryons suggest that the appearance of hybrid cells with a fibroblast-like phenotype reflects the reprogramming, rather than the induced differentiation, of the ES cell genome under the influence of a somatic partner.

Environmental Epigenetics and a Unified Theory of the Molecular Aspects of Evolution: A Neo-Lamarckian Concept that Facilitates Neo-Darwinian Evolution.

PubMed

Skinner, Michael K

2015-04-26

Environment has a critical role in the natural selection process for Darwinian evolution. The primary molecular component currently considered for neo-Darwinian evolution involves genetic alterations and random mutations that generate the phenotypic variation required for natural selection to act. The vast majority of environmental factors cannot directly alter DNA sequence. Epigenetic mechanisms directly regulate genetic processes and can be dramatically altered by environmental factors. Therefore, environmental epigenetics provides a molecular mechanism to directly alter phenotypic variation generationally. Lamarck proposed in 1802 the concept that environment can directly alter phenotype in a heritable manner. Environmental epigenetics and epigenetic transgenerational inheritance provide molecular mechanisms for this process. Therefore, environment can on a molecular level influence the phenotypic variation directly. The ability of environmental epigenetics to alter phenotypic and genotypic variation directly can significantly impact natural selection. Neo-Lamarckian concept can facilitate neo-Darwinian evolution. A unified theory of evolution is presented to describe the integration of environmental epigenetic and genetic aspects of evolution. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Phenotypic diversification by enhanced genome restructuring after induction of multiple DNA double-strand breaks.

PubMed

Muramoto, Nobuhiko; Oda, Arisa; Tanaka, Hidenori; Nakamura, Takahiro; Kugou, Kazuto; Suda, Kazuki; Kobayashi, Aki; Yoneda, Shiori; Ikeuchi, Akinori; Sugimoto, Hiroki; Kondo, Satoshi; Ohto, Chikara; Shibata, Takehiko; Mitsukawa, Norihiro; Ohta, Kunihiro

2018-05-18

DNA double-strand break (DSB)-mediated genome rearrangements are assumed to provide diverse raw genetic materials enabling accelerated adaptive evolution; however, it remains unclear about the consequences of massive simultaneous DSB formation in cells and their resulting phenotypic impact. Here, we establish an artificial genome-restructuring technology by conditionally introducing multiple genomic DSBs in vivo using a temperature-dependent endonuclease TaqI. Application in yeast and Arabidopsis thaliana generates strains with phenotypes, including improved ethanol production from xylose at higher temperature and increased plant biomass, that are stably inherited to offspring after multiple passages. High-throughput genome resequencing revealed that these strains harbor diverse rearrangements, including copy number variations, translocations in retrotransposons, and direct end-joinings at TaqI-cleavage sites. Furthermore, large-scale rearrangements occur frequently in diploid yeasts (28.1%) and tetraploid plants (46.3%), whereas haploid yeasts and diploid plants undergo minimal rearrangement. This genome-restructuring system (TAQing system) will enable rapid genome breeding and aid genome-evolution studies.

[Analysis of genetics mechanism for the phenotypic diversity in a patient carrying a rare ring chromosome 9].

PubMed

Qin, Shengfang; Wang, Xueyan; Li, Yunxing; Wei, Ping; Chen, Chun; Zeng, Lan

2016-02-01

To explore the genetics mechanism for the phenotypic variability in a patient carrying a rare ring chromosome 9. The karyotype of the patient was analyzed with cytogenetics method. Presence of sex chromosome was confirmed with fluorescence in situ hybridization. The SRY gene was subjected to PCR amplification and direct sequencing. Potential deletion and duplication were detected with array-based comparative genomic hybridization (array-CGH). The karyotype of the patient has comprised 6 types of cell lines containing a ring chromosome 9. The SRY gene sequence was normal. By array-CGH, the patient has carried a hemizygous deletion at 9p24.3-p23 (174 201-9 721 761) encompassing 30 genes from Online Mendelian Inheritance in Man. The phenotypic variability of the 9p deletion syndrome in conjunct with ring chromosome 9 may be attributable to multiple factors including loss of chromosomal material, insufficient dosage of genes, instability of ring chromosome, and pattern of inheritance.

Detecting and targeting mesenchymal-like subpopulations within squamous cell carcinomas

PubMed Central

Montone, Kathleen T; Wang, Li-Ping; Gimotty, Phyllis A; Hammond, Rachel; Diehl, J Alan; Rustgi, Anil K; Lee, John T; Rasanen, Kati; Weinstein, Gregory S

2011-01-01

Curative eradication of all cells within carcinomas is seldom achievable with chemotherapy alone. This limitation may be partially attributable to tumor cell subpopulations with intrinsic resistance to current drugs. Within squamous cell carcinoma (SCC) cell lines, we previously characterized a subpopulation of mesenchymal-like cells displaying phenotypic plasticity and increased resistance to both cytotoxic and targeted agents. These mesenchymal-like (Ecad-lo) cells are separable from epithelial-like (Ecad-hi) cells based on loss of surface E-cadherin and expression of vimentin. Despite their long-term plasticity, both Ecad-lo and Ecad-hi subsets in short-term culture maintained nearly uniform phenotypes after purification. This stability allowed testing of segregated subpopulations for relative sensitivity to the cytotoxic agent cisplatin in comparison to salinomycin, a compound with reported activity against CD44+CD24− stem-like cells in breast carcinomas. Salinomycin showed comparable efficacy against both Ecad-hi and Ecad-lo cells in contrast to cisplatin, which selectively depleted Ecad-hi cells. An in vivo correlate of these mesenchymal-like Ecad-lo cells was identified by immunohistochemical detection of vimentin-positive malignant subsets across a part of direct tumor xenografts (DTXs) of advanced stage SCC patient samples. Cisplatin treatment of mice with established DTXs caused enrichment of vimentin-positive malignant cells in residual tumors, but salinomycin depleted the same subpopulation. These results demonstrate that mesenchymal-like SCC cells, which resist current chemotherapies, respond to a treatment strategy developed against a stem-like subset in breast carcinoma. Further, they provide evidence of mesenchymal-like subsets being well-represented across advanced stage SCCs, suggesting that intrinsic drug resistance in this subpopulation has high clinical relevance. PMID:21558812

Detecting and targeting mesenchymal-like subpopulations within squamous cell carcinomas.

PubMed

Basu, Devraj; Montone, Kathleen T; Wang, Li-Ping; Gimotty, Phyllis A; Hammond, Rachel; Diehl, J Alan; Rustgi, Anil K; Lee, John T; Rasanen, Kati; Weinstein, Gregory S; Herlyn, Meenhard

2011-06-15

Curative eradication of all cells within carcinomas is seldom achievable with chemotherapy alone. This limitation may be partially attributable to tumor cell subpopulations with intrinsic resistance to current drugs. Within squamous cell carcinoma (SCC) cell lines, we previously characterized a subpopulation of mesenchymal-like cells displaying phenotypic plasticity and increased resistance to both cytotoxic and targeted agents. These mesenchymal-like (Ecad-lo) cells are separable from epithelial-like (Ecad-hi) cells based on loss of surface E-cadherin and expression of vimentin. Despite their long-term plasticity, both Ecad-lo and Ecad-hi subsets in short-term culture maintained nearly uniform phenotypes after purification. This stability allowed testing of segregated subpopulations for relative sensitivity to the cytotoxic agent cisplatin in comparison to salinomycin, a compound with reported activity against CD44(+)CD24(-) stem-like cells in breast carcinomas. Salinomycin showed comparable efficacy against both Ecad-hi and Ecad-lo cells in contrast to cisplatin, which selectively depleted Ecad-hi cells. An in vivo correlate of these mesenchymal-like Ecad-lo cells was identified by immunohistochemical detection of vimentin-positive malignant subsets across a part of direct tumor xenografts (DTXs) of advanced stage SCC patient samples. Cisplatin treatment of mice with established DTXs caused enrichment of vimentin-positive malignant cells in residual tumors, but salinomycin depleted the same subpopulation. These results demonstrate that mesenchymal-like SCC cells, which resist current chemotherapies, respond to a treatment strategy developed against a stem-like subset in breast carcinoma. Further, they provide evidence of mesenchymal-like subsets being well-represented across advanced stage SCCs, suggesting that intrinsic drug resistance in this subpopulation has high clinical relevance.

Endogenous molecular network reveals two mechanisms of heterogeneity within gastric cancer.

PubMed

Li, Site; Zhu, Xiaomei; Liu, Bingya; Wang, Gaowei; Ao, Ping

2015-05-30

Intratumor heterogeneity is a common phenomenon and impedes cancer therapy and research. Gastric cancer (GC) cells have generally been classified into two heterogeneous cellular phenotypes, the gastric and intestinal types, yet the mechanisms of maintaining two phenotypes and controlling phenotypic transition are largely unknown. A qualitative systematic framework, the endogenous molecular network hypothesis, has recently been proposed to understand cancer genesis and progression. Here, a minimal network corresponding to such framework was found for GC and was quantified via a stochastic nonlinear dynamical system. We then further extended the framework to address the important question of intratumor heterogeneity quantitatively. The working network characterized main known features of normal gastric epithelial and GC cell phenotypes. Our results demonstrated that four positive feedback loops in the network are critical for GC cell phenotypes. Moreover, two mechanisms that contribute to GC cell heterogeneity were identified: particular positive feedback loops are responsible for the maintenance of intestinal and gastric phenotypes; GC cell progression routes that were revealed by the dynamical behaviors of individual key components are heterogeneous. In this work, we constructed an endogenous molecular network of GC that can be expanded in the future and would broaden the known mechanisms of intratumor heterogeneity.

Endogenous molecular network reveals two mechanisms of heterogeneity within gastric cancer

PubMed Central

Li, Site; Zhu, Xiaomei; Liu, Bingya; Wang, Gaowei; Ao, Ping

2015-01-01

Intratumor heterogeneity is a common phenomenon and impedes cancer therapy and research. Gastric cancer (GC) cells have generally been classified into two heterogeneous cellular phenotypes, the gastric and intestinal types, yet the mechanisms of maintaining two phenotypes and controlling phenotypic transition are largely unknown. A qualitative systematic framework, the endogenous molecular network hypothesis, has recently been proposed to understand cancer genesis and progression. Here, a minimal network corresponding to such framework was found for GC and was quantified via a stochastic nonlinear dynamical system. We then further extended the framework to address the important question of intratumor heterogeneity quantitatively. The working network characterized main known features of normal gastric epithelial and GC cell phenotypes. Our results demonstrated that four positive feedback loops in the network are critical for GC cell phenotypes. Moreover, two mechanisms that contribute to GC cell heterogeneity were identified: particular positive feedback loops are responsible for the maintenance of intestinal and gastric phenotypes; GC cell progression routes that were revealed by the dynamical behaviors of individual key components are heterogeneous. In this work, we constructed an endogenous molecular network of GC that can be expanded in the future and would broaden the known mechanisms of intratumor heterogeneity. PMID:25962957

Airway-Resident Memory CD8 T Cells Provide Antigen-Specific Protection against Respiratory Virus Challenge through Rapid IFN-γ Production.

PubMed

McMaster, Sean R; Wilson, Jarad J; Wang, Hong; Kohlmeier, Jacob E

2015-07-01

CD8 airway resident memory T (TRM) cells are a distinctive TRM population with a high turnover rate and a unique phenotype influenced by their localization within the airways. Their role in mediating protective immunity to respiratory pathogens, although suggested by many studies, has not been directly proven. This study provides definitive evidence that airway CD8 TRM cells are sufficient to mediate protection against respiratory virus challenge. Despite being poorly cytolytic in vivo and failing to expand after encountering Ag, airway CD8 TRM cells rapidly express effector cytokines, with IFN-γ being produced most robustly. Notably, established airway CD8 TRM cells possess the ability to produce IFN-γ faster than systemic effector memory CD8 T cells. Furthermore, naive mice receiving intratracheal transfer of airway CD8 TRM cells lacking the ability to produce IFN-γ were less effective at controlling pathogen load upon heterologous challenge. This direct evidence of airway CD8 TRM cell-mediated protection demonstrates the importance of these cells as a first line of defense for optimal immunity against respiratory pathogens and suggests they should be considered in the development of future cell-mediated vaccines. Copyright © 2015 by The American Association of Immunologists, Inc.

Phenotypic Characterization of Retinoic Acid Differentiated SH-SY5Y Cells by Transcriptional Profiling

PubMed Central

Korecka, Joanna A.; van Kesteren, Ronald E.; Blaas, Eva; Spitzer, Sonia O.; Kamstra, Jorke H.; Smit, August B.; Swaab, Dick F.; Verhaagen, Joost; Bossers, Koen

2013-01-01

Multiple genetic and environmental factors play a role in the development and progression of Parkinson’s disease (PD). The main neuropathological hallmark of PD is the degeneration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta. To study genetic and molecular contributors to the disease process, there is a great need for readily accessible cells with prominent DAergic features that can be used for reproducible in vitro cellular screening. Here, we investigated the molecular phenotype of retinoic acid (RA) differentiated SH-SY5Y cells using genome wide transcriptional profiling combined with gene ontology, transcription factor and molecular pathway analysis. We demonstrated that RA induces a general neuronal differentiation program in SH-SY5Y cells and that these cells develop a predominantly mature DAergic-like neurotransmitter phenotype. This phenotype is characterized by increased dopamine levels together with a substantial suppression of other neurotransmitter phenotypes, such as those for noradrenaline, acetylcholine, glutamate, serotonin and histamine. In addition, we show that RA differentiated SH-SY5Y cells express the dopamine and noradrenalin neurotransmitter transporters that are responsible for uptake of MPP(+), a well known DAergic cell toxicant. MPP(+) treatment alters mitochondrial activity according to its proposed cytotoxic effect in DAergic neurons. Taken together, RA differentiated SH-SY5Y cells have a DAergic-like phenotype, and provide a good cellular screening tool to find novel genes or compounds that affect cytotoxic processes that are associated with PD. PMID:23724009

Model-Based Phenotypic Signatures Governing the Dynamics of the Stem and Semi-differentiated Cell Populations in Dysplastic Colonic Crypts.

PubMed

Nikolov, Svetoslav; Santos, Guido; Wolkenhauer, Olaf; Vera, Julio

2018-02-01

Mathematical modeling of cell differentiated in colonic crypts can contribute to a better understanding of basic mechanisms underlying colonic tissue organization, but also its deregulation during carcinogenesis and tumor progression. Here, we combined bifurcation analysis to assess the effect that time delay has in the complex interplay of stem cells and semi-differentiated cells at the niche of colonic crypts, and systematic model perturbation and simulation to find model-based phenotypes linked to cancer progression. The models suggest that stem cell and semi-differentiated cell population dynamics in colonic crypts can display chaotic behavior. In addition, we found that clinical profiling of colorectal cancer correlates with the in silico phenotypes proposed by the mathematical model. Further, potential therapeutic targets for chemotherapy resistant phenotypes are proposed, which in any case will require experimental validation.

In situ macrophage phenotypic transition is affected by altered cellular composition prior to acute sterile muscle injury.

PubMed

Patsalos, Andreas; Pap, Attila; Varga, Tamas; Trencsenyi, Gyorgy; Contreras, Gerardo Alvarado; Garai, Ildiko; Papp, Zoltan; Dezso, Balazs; Pintye, Eva; Nagy, Laszlo

2017-09-01

The in situ phenotypic switch of macrophages is delayed in acute injury following irradiation. The combination of bone marrow transplantation and local muscle radiation protection allows for the identification of a myeloid cell contribution to tissue repair. PET-MRI allows monitoring of myeloid cell invasion and metabolism. Altered cellular composition prior to acute sterile injury affects the in situ phenotypic transition of invading myeloid cells to repair macrophages. There is reciprocal intercellular communication between local muscle cell compartments, such as PAX7 positive cells, and recruited macrophages during skeletal muscle regeneration. Skeletal muscle regeneration is a complex interplay between various cell types including invading macrophages. Their recruitment to damaged tissues upon acute sterile injuries is necessary for clearance of necrotic debris and for coordination of tissue regeneration. This highly dynamic process is characterized by an in situ transition of infiltrating monocytes from an inflammatory (Ly6C high ) to a repair (Ly6C low ) macrophage phenotype. The importance of the macrophage phenotypic shift and the cross-talk of the local muscle tissue with the infiltrating macrophages during tissue regeneration upon injury are not fully understood and their study lacks adequate methodology. Here, using an acute sterile skeletal muscle injury model combined with irradiation, bone marrow transplantation and in vivo imaging, we show that preserved muscle integrity and cell composition prior to the injury is necessary for the repair macrophage phenotypic transition and subsequently for proper and complete tissue regeneration. Importantly, by using a model of in vivo ablation of PAX7 positive cells, we show that this radiosensitive skeletal muscle progenitor pool contributes to macrophage phenotypic transition following acute sterile muscle injury. In addition, local muscle tissue radioprotection by lead shielding during irradiation preserves normal macrophage transition dynamics and subsequently muscle tissue regeneration. Taken together, our data suggest the existence of a more extensive and reciprocal cross-talk between muscle tissue compartments, including satellite cells, and infiltrating myeloid cells upon tissue damage. These interactions shape the macrophage in situ phenotypic shift, which is indispensable for normal muscle tissue repair dynamics. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Phenotypic heterogeneity in metabolic traits among single cells of a rare bacterial species in its natural environment quantified with a combination of flow cell sorting and NanoSIMS

PubMed Central

Zimmermann, Matthias; Escrig, Stéphane; Hübschmann, Thomas; Kirf, Mathias K.; Brand, Andreas; Inglis, R. Fredrik; Musat, Niculina; Müller, Susann; Meibom, Anders; Ackermann, Martin; Schreiber, Frank

2015-01-01

Populations of genetically identical microorganisms residing in the same environment can display marked variability in their phenotypic traits; this phenomenon is termed phenotypic heterogeneity. The relevance of such heterogeneity in natural habitats is unknown, because phenotypic characterization of a sufficient number of single cells of the same species in complex microbial communities is technically difficult. We report a procedure that allows to measure phenotypic heterogeneity in bacterial populations from natural environments, and use it to analyze N2 and CO2 fixation of single cells of the green sulfur bacterium Chlorobium phaeobacteroides from the meromictic lake Lago di Cadagno. We incubated lake water with 15N2 and 13CO2 under in situ conditions with and without NH4+. Subsequently, we used flow cell sorting with auto-fluorescence gating based on a pure culture isolate to concentrate C. phaeobacteroides from its natural abundance of 0.2% to now 26.5% of total bacteria. C. phaeobacteroides cells were identified using catalyzed-reporter deposition fluorescence in situ hybridization (CARD-FISH) targeting the 16S rRNA in the sorted population with a species-specific probe. In a last step, we used nanometer-scale secondary ion mass spectrometry to measure the incorporation 15N and 13C stable isotopes in more than 252 cells. We found that C. phaeobacteroides fixes N2 in the absence of NH4+, but not in the presence of NH4+ as has previously been suggested. N2 and CO2 fixation were heterogeneous among cells and positively correlated indicating that N2 and CO2 fixation activity interact and positively facilitate each other in individual cells. However, because CARD-FISH identification cannot detect genetic variability among cells of the same species, we cannot exclude genetic variability as a source for phenotypic heterogeneity in this natural population. Our study demonstrates the technical feasibility of measuring phenotypic heterogeneity in a rare bacterial species in its natural habitat, thus opening the door to study the occurrence and relevance of phenotypic heterogeneity in nature. PMID:25932020

Ascites-induced shift along epithelial-mesenchymal spectrum in ovarian cancer cells: enhancement of their invasive behavior partly dependant on αv integrins.

PubMed

Carduner, L; Leroy-Dudal, J; Picot, C R; Gallet, O; Carreiras, F; Kellouche, S

2014-08-01

At least one-third of patients with epithelial ovarian cancer (OC) present ascites at diagnosis and almost all have ascites at recurrence. The presence of ascites, which acts as a dynamic reservoir of active molecules and cellular components, correlates with the OC peritoneal metastasis and is associated with poor prognosis. Since epithelial-mesenchymal transition (EMT) is involved in different phases of OC progression, we have investigated the effect of the unique ascitic tumor microenvironment on the EMT status and the behavior of OC cells. The exposure of three OC cell lines to ascites leads to changes in cellular morphologies. Within ascites, OC cells harboring an initial intermediate epithelial phenotype are characterized by marked dislocation of epithelial markers (E-cadherin, ZO-1 staining) while OC cells initially harboring an intermediate mesenchymal phenotype strengthen their mesenchymal markers (N-cadherin, vimentin). Ascites differentially triggers a dissemination phenotype related to the initial cell features by either allowing the proliferation and the formation of spheroids and the extension of colonies for cells that present an initial epithelial intermediate phenotype, or favoring the migration of cells with a mesenchymal intermediate phenotype. In an ascitic microenvironment, a redeployment of αv integrins into cells was observed and the ascites-induced accentuation of the two different invasive phenotypes (i.e. spheroids formation or migration) was shown to involve αv integrins. Thus, ascites induces a shift toward an unstable intermediate state of the epithelial-mesenchymal spectrum and confers a more aggressive cell behavior that takes on a different pathway based on the initial epithelial-mesenchymal cell features.

Differential Effector Engagement by Oncogenic KRAS. | Office of Cancer Genomics

Cancer.gov

KRAS can bind numerous effector proteins, which activate different downstream signaling events. The best known are RAF, phosphatidylinositide (PI)-3' kinase, and RalGDS families, but many additional direct and indirect effectors have been reported. We have assessed how these effectors contribute to several major phenotypes in a quantitative way, using an arrayed combinatorial siRNA screen in which we knocked down 41 KRAS effectors nodes in 92 cell lines.

Cloning of feline FOXP3 and detection of expression in CD4+CD25+ regulatory T cells

PubMed Central

Lankford, Susan; Petty, Christopher; LaVoy, Alora; Reckling, Stacie; Tompkins, Wayne; Dean, Gregg A.

2008-01-01

Regulatory T cells (Treg) are increased and directly infected by feline immunodeficiency virus (FIV) and likely play a role in other feline autoimmune, neoplastic, and infectious diseases. Phenotypically, Treg are best characterized by surface expression of CD4 and CD25 and intranuclear expression of the forkhead transcription factor Foxp3. Our objective was to clone and sequence feline FOXP3 for the purpose of developing assays to enhance studies of feline Treg. We determined the feline FOXP3 is 1293 nucleotides in length and codes for a protein that shares high homology to other species. A splice variant devoid of exon 2 was also identified. A real-time PCR assay was developed and used to show Foxp3 mRNA expression occurs primarily in CD4+CD25+ T cells. Two cross-reacting antibodies were identified by immunocytochemical staining of HEK293 cells transfected with feline FOXP3. The antibody labeling confirmed the nuclear localization of the protein. A flow cytometric assay was also validated and used to correlate the phenotypic and functional characteristics of feline Treg induced by treatment of lymph node lymphocytes with flagellin or LPS in combination with mitogen or IL2. Together, these studies provide useful tools to further investigate Foxp3 and Tregs in cats. PMID:18180044

A kinetic investigation of interacting, stimulated T cells identifies conditions for rapid functional enhancement, minimal phenotype differentiation, and improved adoptive cell transfer tumor eradication.

PubMed

Zhou, Jing; Bethune, Michael T; Malkova, Natalia; Sutherland, Alexander M; Comin-Anduix, Begonya; Su, Yapeng; Baltimore, David; Ribas, Antoni; Heath, James R

2018-01-01

For adoptive cell transfer (ACT) immunotherapy of tumor-reactive T cells, an effective therapeutic outcome depends upon cell dose, cell expansion in vivo through a minimally differentiated phenotype, long term persistence, and strong cytolytic effector function. An incomplete understanding of the biological coupling between T cell expansion, differentiation, and response to stimulation hinders the co-optimization of these factors. We report on a biophysical investigation of how the short-term kinetics of T cell functional activation, through molecular stimulation and cell-cell interactions, competes with phenotype differentiation. T cells receive molecular stimulation for a few minutes to a few hours in bulk culture. Following this priming period, the cells are then analyzed at the transcriptional level, or isolated as single cells, with continuing molecular stimulation, within microchambers for analysis via 11-plex secreted protein assays. We resolve a rapid feedback mechanism, promoted by T cell-T cell contact interactions, which strongly amplifies T cell functional performance while yielding only minimal phenotype differentiation. When tested in mouse models of ACT, optimally primed T cells lead to complete tumor eradication. A similar kinetic process is identified in CD8+ and CD4+ T cells collected from a patient with metastatic melanoma.

Fibronectin Matrix Polymerization Regulates Smooth Muscle Cell Phenotype through a Rac1 Dependent Mechanism

PubMed Central

Shi, Feng; Long, Xiaochun; Hendershot, Allison; Miano, Joseph M.; Sottile, Jane

2014-01-01

Smooth muscle cells are maintained in a differentiated state in the vessel wall, but can be modulated to a synthetic phenotype following injury. Smooth muscle phenotypic modulation is thought to play an important role in the pathology of vascular occlusive diseases. Phenotypically modulated smooth muscle cells exhibit increased proliferative and migratory properties that accompany the downregulation of smooth muscle cell marker proteins. Extracellular matrix proteins, including fibronectin, can regulate the smooth muscle phenotype when used as adhesive substrates. However, cells produce and organize a 3-dimensional fibrillar extracellular matrix, which can affect cell behavior in distinct ways from the protomeric 2-dimensional matrix proteins that are used as adhesive substrates. We previously showed that the deposition/polymerization of fibronectin into the extracellular matrix can regulate the deposition and organization of other extracellular matrix molecules in vitro. Further, our published data show that the presence of a fibronectin polymerization inhibitor results in increased expression of smooth muscle cell differentiation proteins and inhibits vascular remodeling in vivo. In this manuscript, we used an in vitro cell culture system to determine the mechanism by which fibronectin polymerization affects smooth muscle phenotypic modulation. Our data show that fibronectin polymerization decreases the mRNA levels of multiple smooth muscle differentiation genes, and downregulates the levels of smooth muscle α-actin and calponin proteins by a Rac1-dependent mechanism. The expression of smooth muscle genes is transcriptionally regulated by fibronectin polymerization, as evidenced by the increased activity of luciferase reporter constructs in the presence of a fibronectin polymerization inhibitor. Fibronectin polymerization also promotes smooth muscle cell growth, and decreases the levels of actin stress fibers. These data define a Rac1-dependent pathway wherein fibronectin polymerization promotes the SMC synthetic phenotype by modulating the expression of smooth muscle cell differentiation proteins. PMID:24752318

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takahashi, Atsushi; Graduate School of Science and Engineering, Saitama University, Saitama 338-8570; Green Tea Laboratory, Saitama Prefectural Agriculture and Forestry Research Center, Saitama 358-0042

Highlights: •EGCG reduced cell motility of highly metastatic human lung cancer cells. •EGCG increased cell stiffness of the cells, indicating the inhibition of phenotypes of EMT. •EGCG inhibited expression of vimentin and Slug in the cells at the leading edge of scratch. •Treatment of MβCD increased cell stiffness, and inhibited cell motility and vimentin expression. •Inhibition of EMT phenotypes with EGCG is a mechanism-based inhibition of cancer metastasis. -- Abstract: Cell motility and cell stiffness are closely related to metastatic activity of cancer cells. (−)-Epigallocatechin gallate (EGCG) has been shown to inhibit spontaneous metastasis of melanoma cell line into themore » lungs of mice, so we studied the effects of EGCG on cell motility, cell stiffness, and expression of vimentin and Slug, which are molecular phenotypes of epithelial–mesenchymal transition (EMT). Treatments of human non-small cell lung cancer cell lines H1299 and Lu99 with 50 and 100 μM EGCG reduced cell motility to 67.5% and 43.7% in H1299, and 71.7% and 31.5% in Lu99, respectively in in vitro wound healing assay. Studies on cell stiffness using atomic force microscope (AFM) revealed that treatment with 50 μM EGCG increased Young’s modulus of H1299 from 1.24 to 2.25 kPa and that of Lu99 from 1.29 to 2.28 kPa, showing a 2-fold increase in cell stiffness, i.e. rigid elasticity of cell membrane. Furthermore, treatment with 50 μM EGCG inhibited high expression of vimentin and Slug in the cells at a leading edge of scratch. Methyl-β-cyclodextrin, a reagent to deplete cholesterol in plasma membrane, showed inhibition of EMT phenotypes similar that by EGCG, suggesting that EGCG induces inhibition of EMT phenotypes by alteration of membrane organization.« less

Endothelial and Epithelial Cell Transition to a Mesenchymal Phenotype Was Delineated by Nestin Expression.

PubMed

Chabot, Andréanne; Hertig, Vanessa; Boscher, Elena; Nguyen, Quang Trinh; Boivin, Benoît; Chebli, Jasmine; Bissonnette, Lyse; Villeneuve, Louis; Brochiero, Emmanuelle; Dupuis, Jocelyn; Calderone, Angelino

2016-07-01

Endothelial and epithelial cell transition to a mesenchymal phenotype was identified as cellular paradigms implicated in the appearance of fibroblasts and development of reactive fibrosis in interstitial lung disease. The intermediate filament protein nestin was highly expressed in fibrotic tissue, detected in fibroblasts and participated in proliferation and migration. The present study tested the hypothesis that the transition of endothelial and epithelial cells to a mesenchymal phenotype was delineated by nestin expression. Three weeks following hypobaric hypoxia, adult male Sprague-Dawley rats characterized by alveolar and perivascular lung fibrosis were associated with increased nestin protein and mRNA levels and marked appearance of nestin/collagen type I((+))-fibroblasts. In the perivascular region of hypobaric hypoxic rats, displaced CD31((+))-endothelial cells were detected, exhibited a mesenchymal phenotype and co-expressed nestin. Likewise, epithelial cells in the lungs of hypobaric hypoxic rats transitioned to a mesenchymal phenotype distinguished by the co-expression of E-cadherin and collagen. Following the removal of FBS from primary passage rat alveolar epithelial cells, TGF-β1 was detected in the media and a subpopulation acquired a mesenchymal phenotype characterized by E-cadherin downregulation and concomitant induction of collagen and nestin. Bone morphogenic protein-7 treatment of alveolar epithelial cells prevented E-cadherin downregulation, suppressed collagen induction but partially inhibited nestin expression. These data support the premise that the transition of endothelial and epithelial cells to a mesenchymal cell may have contributed in part to the appearance nestin/collagen type I((+))-fibroblasts and the reactive fibrotic response in the lungs of hypobaric hypoxic rats. © 2015 Wiley Periodicals, Inc.

CellCognition: time-resolved phenotype annotation in high-throughput live cell imaging.

PubMed

Held, Michael; Schmitz, Michael H A; Fischer, Bernd; Walter, Thomas; Neumann, Beate; Olma, Michael H; Peter, Matthias; Ellenberg, Jan; Gerlich, Daniel W

2010-09-01

Fluorescence time-lapse imaging has become a powerful tool to investigate complex dynamic processes such as cell division or intracellular trafficking. Automated microscopes generate time-resolved imaging data at high throughput, yet tools for quantification of large-scale movie data are largely missing. Here we present CellCognition, a computational framework to annotate complex cellular dynamics. We developed a machine-learning method that combines state-of-the-art classification with hidden Markov modeling for annotation of the progression through morphologically distinct biological states. Incorporation of time information into the annotation scheme was essential to suppress classification noise at state transitions and confusion between different functional states with similar morphology. We demonstrate generic applicability in different assays and perturbation conditions, including a candidate-based RNA interference screen for regulators of mitotic exit in human cells. CellCognition is published as open source software, enabling live-cell imaging-based screening with assays that directly score cellular dynamics.

In vitro haematopoiesis of a novel dendritic-like cell present in murine spleen.

PubMed

Tan, Jonathan K H; O'Neill, Helen C

2010-12-01

Dendritic cells (DC) are important antigen presenting cells (APC) which induce and control the adaptive immune response. In spleen alone, multiple DC subsets can be distinguished by cell surface marker phenotype. Most of these have been shown to develop from progenitors in bone marrow and to seed lymphoid and tissue sites during development. This study advances in vitro methodology for haematopoiesis of dendritic-like cells from progenitors in spleen. Since spleen progenitors undergo differentiation in vitro to produce these cells, the possibility exists that spleen represents a specific niche for differentiation of this subset. The fact that an equivalent cell subset has been shown to exist in spleen also supports that hypothesis. Studies have been directed at investigating the specific functional role of this novel subset as an APC accessible to blood-borne antigen, as well as the conditions under which haematopoiesis is initiated in spleen, and the type of progenitor involved.

Cytokinesis defect in BY-2 cells caused by ATP-competitive kinase inhibitors.

PubMed

Kozgunova, Elena; Higashiyama, Tetsuya; Kurihara, Daisuke

2016-10-02

Cytokinesis is last but not least in cell division as it completes the formation of the two cells. The main role in cell plate orientation and expansion have been assigned to microtubules and kinesin proteins. However, recently we reported severe cytokinesis defect in BY-2 cells not accompanied by changes in microtubules dynamics. Here we also confirmed that distribution of kinesin NACK1 is not the cause of cytokinesis defect. We further explored inhibition of the cell plate expansion by ATP-competitive inhibitors. Two different inhibitors, 5-Iodotubercidin and ML-7 resulted in a very similar phenotype, which indicates that they target same protein cascade. Interestingly, in our previous study we showed that 5-Iodotubercidin treatment affects concentration of actin filaments on the cell plate, while ML-7 is inhibitor of myosin light chain kinase. Although not directly, it indicates importance of actomyosin complex in plant cytokinesis.

Attachment, invasion, chemotaxis, and proteinase expression of B16-BL6 melanoma cells exhibiting a low metastatic phenotype after exposure to dietary restriction of tyrosine and phenylalanine.

PubMed

Uhlenkott, C E; Huijzer, J C; Cardeiro, D J; Elstad, C A; Meadows, G G

1996-03-01

We previously reported that low levels of tyrosine (Tyr) and phenylalanine (Phe) alter the metastatic phenotype of B16-BL6 (BL6) murine melanoma and select for tumor cell populations with decreased lung colonizing ability. To more specifically characterize the effects of Tyr and Phe restriction on the malignant phenotype of BL6, we investigated in vitro attachment, invasion, proteinase expression, and chemotaxis of high and low metastatic BL6 variants. High metastatic variant cells were isolated from subcutaneous tumors of mice fed a nutritionally complete diet (ND cells) and low metastatic variant cells were isolated from mice fed a diet restricted in Tyr and Phe (LTP cells). Results indicate that attachment to reconstituted basement membrane (Matrigel) was significantly reduced in LTP cells as compared to ND cells. Attachment to collagen IV, laminin, and fibronectin were similar between the two variants. Invasion through Matrigel and growth factor-reduced Matrigel were significantly decreased in LTP cells as compared to ND cells. Zymography revealed the presence of M(r) 92,000 and M(r) 72,000 progelatinases, tissue plasminogen activator, and urokinase plasminogen activator in the conditioned medium of both variants; however, there were no differences in activity of these secreted proteinases between the two variants. Growth of the variants on growth factor-reduced Matrigel similarly induced expression of the M(r) 92,000 progelatinase. The variants exhibited similar chemotactic responses toward laminin. However, the chemotactic response toward fibronectin by LTP cells was significantly increased. MFR5, a monoclonal antibody which selectively blocks function of the alpha 5 chain of the alpha 5 beta 1 integrin, VLA-5, decreased the chemotactic response toward fibronectin of ND cells by 37%; the chemotactic response by LTP cells was reduced by 49%. This effect was specific for fibronectin-mediated chemotaxis since the chemotaxis toward laminin and invasion through Matrigel were not altered by the presence of MFR5. The surface expression of VLA-5 was significantly increased in LTP cells as compared to ND cells by flow cytometric analysis. These observations suggest that limitation of Tyr and Phe either directly modifies BL6 or selects for subpopulations with altered in vitro invasion, chemotaxis, and integrin expression.

LIN28A enhances the therapeutic potential of cultured neural stem cells in a Parkinson's disease model.

PubMed

Rhee, Yong-Hee; Kim, Tae-Ho; Jo, A-Young; Chang, Mi-Yoon; Park, Chang-Hwan; Kim, Sang-Mi; Song, Jae-Jin; Oh, Sang-Min; Yi, Sang-Hoon; Kim, Hyeon Ho; You, Bo-Hyun; Nam, Jin-Wu; Lee, Sang-Hun

2016-10-01

The original properties of tissue-specific stem cells, regardless of their tissue origins, are inevitably altered during in vitro culturing, lessening the clinical and research utility of stem cell cultures. Specifically, neural stem cells derived from the ventral midbrain lose their dopamine neurogenic potential, ventral midbrain-specific phenotypes, and repair capacity during in vitro cell expansion, all of which are critical concerns in using the cultured neural stem cells in therapeutic approaches for Parkinson's disease. In this study, we observed that the culture-dependent changes of neural stem cells derived from the ventral midbrain coincided with loss of RNA-binding protein LIN28A expression. When LIN28A expression was forced and sustained during neural stem cell expansion using an inducible expression-vector system, loss of dopamine neurogenic potential and midbrain phenotypes after long-term culturing was blocked. Furthermore, dopamine neurons that differentiated from neural stem cells exhibited remarkable survival and resistance against toxic insults. The observed effects were not due to a direct action of LIN28A on the differentiated dopamine neurons, but rather its action on precursor neural stem cells as exogene expression was switched off in the differentiating/differentiated cultures. Remarkable and reproducible behavioural recovery was shown in all Parkinson's disease rats grafted with neural stem cells expanded with LIN28A expression, along with extensive engraftment of dopamine neurons expressing mature neuronal and midbrain-specific markers. These findings suggest that LIN28A expression during stem cell expansion could be used to prepare therapeutically competent donor cells. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Tumor-infiltrating Tim-3+ T cells proliferate avidly except when PD-1 is co-expressed: Evidence for intracellular cross talk

PubMed Central

Li, Jing; Shayan, Gulidanna; Avery, Lyndsay; Jie, Hyun-Bae; Gildener-Leapman, Neil; Schmitt, Nicole; Lu, Bin Feng; Kane, Lawrence P.; Ferris, Robert L.

2016-01-01

ABSTRACT Programmed Death 1 (PD-1) and T cell Ig and mucin domain-3 protein (Tim-3) are immune checkpoint receptors highly expressed on tumor infiltrating T lymphocytes (TIL). PD-1 inhibits T cell activation and type-1 T cell responses, while Tim-3 is proposed to mark more extensively exhausted cells, although the mechanisms underlying Tim-3 function are not clear. Trials of anti-PD-1 therapy have identified a large subset of non-responder patients, likely due to expression of alternative checkpoint molecules like Tim-3. We investigated the phenotypic and functional characteristics of T cells with differential expression of PD-1 (high/low) and Tim-3 (positive/negative), using TIL directly isolated from head and neck squamous cell carcinomas (HNSCC). Unexpectedly, we found that expression of Tim-3 alone does not necessarily mark TIL as dysfunctional/exhausted. In Tim-3-TIL, PD-1 levels correlate with T cell dysfunction, with a PD-1low/intermed phenotype identifying recently activated and still functional cells, whereas PD-1hiTim-3− T cells are actually exhausted. Nonetheless, PD-1intermed cells are still potently suppressed by PD-L1. PD-1 expression was associated with reduced phosphorylation of ribosomal protein S6 (pS6), whereas Tim-3 expression was associated with increased pS6. Using a novel mouse model for inducible Tim-3 expression, we confirmed that expression of Tim-3 does not necessarily render T cells refractory to further activation. These results suggest the existence of PD-1 and Tim-3 crosstalk in regulating antitumor T cell responses, with important implications for anti-PD-1 immunotherapy. PMID:27853635

A kinetic investigation of interacting, stimulated T cells identifies conditions for rapid functional enhancement, minimal phenotype differentiation, and improved adoptive cell transfer tumor eradication

PubMed Central

Zhou, Jing; Bethune, Michael T.; Malkova, Natalia; Sutherland, Alexander M.; Comin-Anduix, Begonya; Su, Yapeng; Baltimore, David; Ribas, Antoni

2018-01-01

For adoptive cell transfer (ACT) immunotherapy of tumor-reactive T cells, an effective therapeutic outcome depends upon cell dose, cell expansion in vivo through a minimally differentiated phenotype, long term persistence, and strong cytolytic effector function. An incomplete understanding of the biological coupling between T cell expansion, differentiation, and response to stimulation hinders the co-optimization of these factors. We report on a biophysical investigation of how the short-term kinetics of T cell functional activation, through molecular stimulation and cell-cell interactions, competes with phenotype differentiation. T cells receive molecular stimulation for a few minutes to a few hours in bulk culture. Following this priming period, the cells are then analyzed at the transcriptional level, or isolated as single cells, with continuing molecular stimulation, within microchambers for analysis via 11-plex secreted protein assays. We resolve a rapid feedback mechanism, promoted by T cell—T cell contact interactions, which strongly amplifies T cell functional performance while yielding only minimal phenotype differentiation. When tested in mouse models of ACT, optimally primed T cells lead to complete tumor eradication. A similar kinetic process is identified in CD8+ and CD4+ T cells collected from a patient with metastatic melanoma. PMID:29360859

Association of Immunological Cell Profiles with Specific Clinical Phenotypes of Scleroderma Disease

PubMed Central

Calzada, David; Mayayo, Teodoro; González-Rodríguez, María Luisa; Rabasco, Antonio María; Lahoz, Carlos

2014-01-01

This study aimed to search the correlation among immunological profiles and clinical phenotypes of scleroderma in well-characterized groups of scleroderma patients, comparing forty-nine scleroderma patients stratified according to specific clinical phenotypes with forty-nine healthy controls. Five immunological cell subpopulations (B, CD4+ and CD8+ T-cells, NK, and monocytes) and their respective stages of apoptosis and activation were analyzed by flow cytometry, in samples of peripheral blood mononuclear cells (PBMCs). Analyses of results were stratified according to disease stage, time since the diagnosis, and visceral damage (pulmonary fibrosis, pulmonary hypertension, and cardiac affliction) and by time of treatment with corticosteroids. An increase in the percentages of monocytes and a decrease in the B cells were mainly related to the disease progression. A general apoptosis decrease was found in all phenotypes studied, except in localized scleroderma. An increase of B and NK cells activation was found in patients diagnosed more than 10 years ago. Specific cell populations like monocytes, NK, and B cells were associated with the type of affected organ. This study shows how, in a heterogeneous disease, proper patient's stratification according to clinical phenotypes allows finding specific cellular profiles. Our data may lead to improvements in the knowledge of prognosis factors and to aid in the analysis of future specific therapies. PMID:24818126

Suppression of miR-184 in malignant gliomas upregulates SND1 and promotes tumor aggressiveness

PubMed Central

Emdad, Luni; Janjic, Aleksandar; Alzubi, Mohammad A.; Hu, Bin; Santhekadur, Prasanna K.; Menezes, Mitchell E.; Shen, Xue-Ning; Das, Swadesh K.; Sarkar, Devanand; Fisher, Paul B.

2015-01-01

Background Malignant glioma is an aggressive cancer requiring new therapeutic targets. MicroRNAs (miRNAs) regulate gene expression post transcriptionally and are implicated in cancer development and progression. Deregulated expressions of several miRNAs, specifically hsa-miR-184, correlate with glioma development. Methods Bioinformatic approaches were used to identify potential miR-184-regulated target genes involved in malignant glioma progression. This strategy identified a multifunctional nuclease, SND1, known to be overexpressed in multiple cancers, including breast, colon, and hepatocellular carcinoma, as a putative direct miR-184 target gene. SND1 levels were evaluated in patient tumor samples and human-derived cell lines. We analyzed invasion and signaling in vitro through SND1 gain-of-function and loss-of-function. An orthotopic xenograft model with primary glioma cells demonstrated a role of miR-184/SND1 in glioma pathogenesis in vivo. Results SND1 is highly expressed in human glioma tissue and inversely correlated with miR-184 expression. Transfection of glioma cells with a miR-184 mimic inhibited invasion, suppressed colony formation, and reduced anchorage-independent growth in soft agar. Similar phenotypes were evident when SND1 was knocked down with siRNA. Additionally, knockdown (KD) of SND1 induced senescence and improved the chemoresistant properties of malignant glioma cells. In an orthotopic xenograft model, KD of SND1 or transfection with a miR-184 mimic induced a less invasive tumor phenotype and significantly improved survival of tumor bearing mice. Conclusions Our study is the first to show a novel regulatory role of SND1, a direct target of miR-184, in glioma progression, suggesting that the miR-184/SND1 axis may be a useful diagnostic and therapeutic tool for malignant glioma. PMID:25216670

Excess 25-hydroxyvitamin D3 exacerbates tubulointerstitial injury in mice by modulating macrophage phenotype.

PubMed

Kusunoki, Yasuo; Matsui, Isao; Hamano, Takayuki; Shimomura, Akihiro; Mori, Daisuke; Yonemoto, Sayoko; Takabatake, Yoshitsugu; Tsubakihara, Yoshiharu; St-Arnaud, René; Isaka, Yoshitaka; Rakugi, Hiromi

2015-11-01

Vitamin D hydroxylated at carbon 25 (25(OH)D) is generally recognized as a precursor of active vitamin D. Despite its low affinity for the vitamin D receptor (VDR), both deficient and excessive 25(OH)D levels are associated with poor clinical outcomes. Here we studied direct effects of 25(OH)D3 on the kidney using 25(OH)D-1α-hydroxylase (CYP27B1) knockout mice. The effects of 25(OH)D3 on unilateral ureteral obstruction were analyzed as proximal tubular cells and macrophages are two major cell types that take up 25(OH)D and contribute to the pathogenesis of kidney injury. Excess 25(OH)D3 in obstructed mice worsened oxidative stress and tubulointerstitial fibrosis, whereas moderate levels of 25(OH)D3 had no effects. The exacerbating effects of excess 25(OH)D3 were abolished in CYP27B1/VDR double-knockout mice and in macrophage-depleted CYP27B1 knockout mice. Excess 25(OH)D3 upregulated both M1 marker (TNF-α) and M2 marker (TGF-β1) levels of kidney-infiltrating macrophages. In vitro analyses verified that excess 25(OH)D3 directly upregulated TNF-α and TGF-β1 in cultured macrophages but not in tubular cells. TNF-α and 25(OH)D3 cooperatively induced oxidative stress by upregulating iNOS in tubular cells. Aggravated tubulointerstitial fibrosis in mice with excess 25(OH)D3 indicated that macrophage-derived TGF-β1 also had a key role in the pathogenesis of surplus 25(OH)D3. Thus, excess 25(OH)D3 worsens tubulointerstitial injury by modulating macrophage phenotype.

Mechanism-based facilitated maturation of human pluripotent stem cell-derived cardiomyocytes

PubMed Central

Lieu, Deborah K.; Fu, Ji-Dong; Chiamvimonvat, Nipavan; Chan Tung, Kelvin W.; McNerney, Gregory P.; Huser, Thomas; Keller, Gordon; Kong, Chi-Wing

2013-01-01

Background Human embryonic stem cells (hESCs) can be efficiently and reproducibly directed into cardiomyocytes (CMs) using stage-specific induction protocols. However, their functional properties and suitability for clinical and other applications have not been evaluated. Methods and Results Here we showed that CMs derived from multiple pluripotent human stem cell lines (hESC: H1, HES2) and types (induced pluripotent stem cell or iPSC) using different in vitro differentiation protocols (embryoid body formation, endodermal induction, directed differentiation) commonly displayed immature, pro-arrhythmic action potential (AP) properties such as high-degree of automaticity, depolarized resting membrane potential (RMP), Phase 4- depolarization and delayed after-depolarization (DAD). Among the panoply of sarcolemmal ionic currents investigated (INa+/ICaL2+/IKr+/INCX+/If+/Ito+/IK1-/IKs-), we pinpointed the lack of the Kir2.1-encoded inwardly rectifying K+ current (IK1) as the single mechanistic contributor to the observed immature electrophysiological properties in hESC-CMs. Forced expression of Kir2.1 in hESC-CMs led to robust expression of Ba2+-sensitive IK1 and more importantly, completely ablated all the pro-arrhythmic AP traits, rendering the electrophysiological phenotype indistinguishable from the adult counterparts. These results provided the first link of a complex developmentally arrested phenotype to a major effector gene, and importantly, further led us to develop a biomimetic culturing strategy for enhancing maturation. Conclusions By providing the environmental cues that are missing in conventional culturing method, this approach did not require any genetic or pharmacological interventions. Our findings can facilitate clinical applications, drug discovery and cardiotoxicity screening by improving the yield, safety and efficacy of derived CMs. PMID:23392582

Macrophage Phenotypes Regulate Scar Formation and Chronic Wound Healing.

PubMed

Hesketh, Mark; Sahin, Katherine B; West, Zoe E; Murray, Rachael Z

2017-07-17

Macrophages and inflammation play a beneficial role during wound repair with macrophages regulating a wide range of processes, such as removal of dead cells, debris and pathogens, through to extracellular matrix deposition re-vascularisation and wound re-epithelialisation. To perform this range of functions, these cells develop distinct phenotypes over the course of wound healing. They can present with a pro-inflammatory M1 phenotype, more often found in the early stages of repair, through to anti-inflammatory M2 phenotypes that are pro-repair in the latter stages of wound healing. There is a continuum of phenotypes between these ranges with some cells sharing phenotypes of both M1 and M2 macrophages. One of the less pleasant consequences of quick closure, namely the replacement with scar tissue, is also regulated by macrophages, through their promotion of fibroblast proliferation, myofibroblast differentiation and collagen deposition. Alterations in macrophage number and phenotype disrupt this process and can dictate the level of scar formation. It is also clear that dysregulated inflammation and altered macrophage phenotypes are responsible for hindering closure of chronic wounds. The review will discuss our current knowledge of macrophage phenotype on the repair process and how alterations in the phenotypes might alter wound closure and the final repair quality.

Allele-specific RNA interference rescues the long-QT syndrome phenotype in human-induced pluripotency stem cell cardiomyocytes.

PubMed

Matsa, Elena; Dixon, James E; Medway, Christopher; Georgiou, Orestis; Patel, Minal J; Morgan, Kevin; Kemp, Paul J; Staniforth, Andrew; Mellor, Ian; Denning, Chris

2014-04-01

Long-QT syndromes (LQTS) are mostly autosomal-dominant congenital disorders associated with a 1:1000 mutation frequency, cardiac arrest, and sudden death. We sought to use cardiomyocytes derived from human-induced pluripotency stem cells (hiPSCs) as an in vitro model to develop and evaluate gene-based therapeutics for the treatment of LQTS. We produced LQTS-type 2 (LQT2) hiPSC cardiomyocytes carrying a KCNH2 c.G1681A mutation in a IKr ion-channel pore, which caused impaired glycosylation and channel transport to cell surface. Allele-specific RNA interference (RNAi) directed towards the mutated KCNH2 mRNA caused knockdown, while leaving the wild-type mRNA unaffected. Electrophysiological analysis of patient-derived LQT2 hiPSC cardiomyocytes treated with mutation-specific siRNAs showed normalized action potential durations (APDs) and K(+) currents with the concurrent rescue of spontaneous and drug-induced arrhythmias (presented as early-afterdepolarizations). These findings provide in vitro evidence that allele-specific RNAi can rescue diseased phenotype in LQTS cardiomyocytes. This is a potentially novel route for the treatment of many autosomal-dominant-negative disorders, including those of the heart.

Chemical genetic screening identifies a novel inhibitor of parallel alignment of cortical microtubules and cellulose microfibrils.

PubMed

Yoneda, Arata; Higaki, Takumi; Kutsuna, Natsumaro; Kondo, Yoichi; Osada, Hiroyuki; Hasezawa, Seiichiro; Matsui, Minami

2007-10-01

It is a well-known hypothesis that cortical microtubules control the direction of cellulose microfibril deposition, and that the parallel cellulose microfibrils determine anisotropic cell expansion and plant cell morphogenesis. However, the molecular mechanism by which cortical microtubules regulate the orientation of cellulose microfibrils is still unclear. To investigate this mechanism, chemical genetic screening was performed. From this screening, 'SS compounds' were identified that induced a spherical swelling phenotype in tobacco BY-2 cells. The SS compounds could be categorized into three classes: those that disrupted the cortical microtubules; those that reduced cellulose microfibril content; and thirdly those that had neither of these effects. In the last class, a chemical designated 'cobtorin' was found to induce the spherical swelling phenotype at the lowest concentration, suggesting strong binding activity to the putative target. Examining cellulose microfibril regeneration using taxol-treated protoplasts revealed that the cobtorin compound perturbed the parallel alignment of pre-existing cortical microtubules and nascent cellulose microfibrils. Thus, cobtorin could be a novel inhibitor and an attractive tool for further investigation of the mechanism that enables cortical microtubules to guide the parallel deposition of cellulose microfibrils.

Mycobacterium avium subspecies impair dendritic cell maturation.

PubMed

Basler, Tina; Brumshagen, Christina; Beineke, Andreas; Goethe, Ralph; Bäumer, Wolfgang

2013-10-01

Mycobacterium avium ssp. paratuberculosis (MAP) causes Johne's disease, a chronic, granulomatous enteritis of ruminants. Dendritic cells (DC) of the gut are ideally placed to combat invading mycobacteria; however, little is known about their interaction with MAP. Here, we investigated the interaction of MAP and the closely related M. avium ssp. avium (MAA) with murine DC and the effect of infected macrophages on DC maturation. The infection of DC with MAP or MAA induced DC maturation, which differed to that of LPS as maturation was accompanied by higher production of IL-10 and lower production of IL-12. Treatment of maturing DC with supernatants from mycobacteria-infected macrophages resulted in impaired DC maturation, leading to a semi-mature, tolerogenic DC phenotype expressing low levels of MHCII, CD86 and TNF-α after LPS stimulation. Though the cells were not completely differentiated they responded with an increased IL-10 and a decreased IL-12 production. Using recombinant cytokines we provide evidence that the semi-mature DC phenotype results from a combination of secreted cytokines and released antigenic mycobacterial components of the infected macrophage. Our results indicate that MAP and MAA are able to subvert DC function directly by infecting and indirectly via the milieu created by infected macrophages.

Allele-specific RNA interference rescues the long-QT syndrome phenotype in human-induced pluripotency stem cell cardiomyocytes

PubMed Central

Matsa, Elena; Dixon, James E.; Medway, Christopher; Georgiou, Orestis; Patel, Minal J.; Morgan, Kevin; Kemp, Paul J.; Staniforth, Andrew; Mellor, Ian; Denning, Chris

2014-01-01

Aims Long-QT syndromes (LQTS) are mostly autosomal-dominant congenital disorders associated with a 1:1000 mutation frequency, cardiac arrest, and sudden death. We sought to use cardiomyocytes derived from human-induced pluripotency stem cells (hiPSCs) as an in vitro model to develop and evaluate gene-based therapeutics for the treatment of LQTS. Methods and results We produced LQTS-type 2 (LQT2) hiPSC cardiomyocytes carrying a KCNH2 c.G1681A mutation in a IKr ion-channel pore, which caused impaired glycosylation and channel transport to cell surface. Allele-specific RNA interference (RNAi) directed towards the mutated KCNH2 mRNA caused knockdown, while leaving the wild-type mRNA unaffected. Electrophysiological analysis of patient-derived LQT2 hiPSC cardiomyocytes treated with mutation-specific siRNAs showed normalized action potential durations (APDs) and K+ currents with the concurrent rescue of spontaneous and drug-induced arrhythmias (presented as early-afterdepolarizations). Conclusions These findings provide in vitro evidence that allele-specific RNAi can rescue diseased phenotype in LQTS cardiomyocytes. This is a potentially novel route for the treatment of many autosomal-dominant-negative disorders, including those of the heart. PMID:23470493

Cancer biomarker discovery: the entropic hallmark.

PubMed

Berretta, Regina; Moscato, Pablo

2010-08-18

It is a commonly accepted belief that cancer cells modify their transcriptional state during the progression of the disease. We propose that the progression of cancer cells towards malignant phenotypes can be efficiently tracked using high-throughput technologies that follow the gradual changes observed in the gene expression profiles by employing Shannon's mathematical theory of communication. Methods based on Information Theory can then quantify the divergence of cancer cells' transcriptional profiles from those of normally appearing cells of the originating tissues. The relevance of the proposed methods can be evaluated using microarray datasets available in the public domain but the method is in principle applicable to other high-throughput methods. Using melanoma and prostate cancer datasets we illustrate how it is possible to employ Shannon Entropy and the Jensen-Shannon divergence to trace the transcriptional changes progression of the disease. We establish how the variations of these two measures correlate with established biomarkers of cancer progression. The Information Theory measures allow us to identify novel biomarkers for both progressive and relatively more sudden transcriptional changes leading to malignant phenotypes. At the same time, the methodology was able to validate a large number of genes and processes that seem to be implicated in the progression of melanoma and prostate cancer. We thus present a quantitative guiding rule, a new unifying hallmark of cancer: the cancer cell's transcriptome changes lead to measurable observed transitions of Normalized Shannon Entropy values (as measured by high-throughput technologies). At the same time, tumor cells increment their divergence from the normal tissue profile increasing their disorder via creation of states that we might not directly measure. This unifying hallmark allows, via the the Jensen-Shannon divergence, to identify the arrow of time of the processes from the gene expression profiles, and helps to map the phenotypical and molecular hallmarks of specific cancer subtypes. The deep mathematical basis of the approach allows us to suggest that this principle is, hopefully, of general applicability for other diseases.